A decellularized small porcine liver.
Jennifer Cisneros was 18 years old, commuting to college from her family's home outside Annapolis, Maryland, when she came down with what she thought was the flu. Over the following weeks, however, her fatigue and nausea worsened, and her weight began to plummet. Alarmed, her mother took her to see a pediatrician. "When I came back with the urine cup, it was orange," Cisneros recalls. "He was like, 'Oh, my God. I've got to send you for blood work.'"
"Eventually, we'll be better off than with a human organ."
Further tests showed that her kidneys were failing, and at Johns Hopkins Hospital, a biopsy revealed the cause: Goodpasture syndrome (GPS), a rare autoimmune disease that attacks the kidneys or lungs. Cisneros was put on dialysis to filter out the waste products that her body could no longer process, and given chemotherapy and steroids to suppress her haywire immune system.
The treatment drove her GPS into remission, but her kidneys were beyond saving. At 19, Cisneros received a transplant, with her mother as donor. Soon, she'd recovered enough to return to school; she did some traveling, and even took up skydiving and parasailing. Then, after less than two years, rejection set in, and the kidney had to be removed.
She went back on dialysis until she was 26, when a stranger learned of her plight and volunteered to donate. That kidney lasted four years, but gave out after a viral infection. Since 2015, Cisneros—now 32, and working as an office administrator between thrice-weekly blood-filtering sessions—has been waiting for a replacement.
She's got plenty of company. About 116,000 people in the United States currently need organ transplants, but fewer than 35,000 organs become available every year. On average, 20 people on the waiting list die each day. And despite repeated campaigns to boost donorship, the gap shows no sign of narrowing.
"This is going to revolutionize medicine, in ways we probably can't yet appreciate."
For decades, doctors and scientists have envisioned a radical solution to the shortage: harvesting other species for spare parts. Xenotransplantation, as the practice is known, could provide an unlimited supply of lifesaving organs for patients like Cisneros. Those organs, moreover, could be altered by genetic engineering or other methods to reduce the danger of rejection—and thus to eliminate the need for immunosuppressive drugs, whose potential side effects include infections, diabetes, and cancer. "Eventually, we'll be better off than with a human organ," says David Cooper, MD, PhD, co-director of the xenotransplant program at the University of Alabama School of Medicine. "This is going to revolutionize medicine, in ways we probably can't yet appreciate."
Recently, progress toward that revolution has accelerated sharply. The cascade of advances began in April 2016, when researchers at the National Heart, Lung, and Blood Institute (NHLBI) reported keeping pig hearts beating in the abdomens of five baboons for a record-breaking mean of 433 days, with one lasting more than two-and-a-half years. Then a team at Emory University announced that a pig kidney sustained a rhesus monkey for 435 days before being rejected, nearly doubling the previous record. At the University of Munich, in Germany, researchers doubled the record for a life-sustaining pig heart transplant in a baboon (replacing the animal's own heart) to 90 days. Investigators at the Salk Institute and the University of California, Davis, declared that they'd grown tissue in pig embryos using human stem cells—a first step toward cultivating personalized replacement organs. The list goes on.
Such breakthroughs, along with a surge of cash from biotech investors, have propelled a wave of bullish media coverage. Yet this isn't the first time that xenotransplantation has been touted as the next big thing. Twenty years ago, the field seemed poised to overcome its final hurdles, only to encounter a setback from which it is just now recovering.
Which raises a question: Is the current excitement justified? Or is the hype again outrunning the science?
A History of Setbacks
The idea behind xenotransplantation dates back at least as far as the 17th century, when French physician Jean-Baptiste Denys tapped the veins of sheep and cows to perform the first documented human blood transfusions. (The practice was banned after two of the four patients died, probably from an immune reaction.) In the 19th century, surgeons began transplanting corneas from pigs and other animals into humans, and using skin xenografts to aid in wound healing; despite claims of miraculous cures, medical historians believe those efforts were mostly futile. In the 1920s and '30s, thousands of men sought renewed vigor through testicular implants from monkeys or goats, but the fad collapsed after studies showed the effects to be imaginary.
Research shut down when scientists discovered a virus in pig DNA that could infect human cells.
After the first successful human organ transplant in 1954—of a kidney, passed between identical twin sisters—the limited supply of donor organs brought a resurgence of interest in animal sources. Attention focused on nonhuman primates, our species' closest evolutionary relatives. At Tulane University, surgeon Keith Reemstma performed the first chimpanzee-to-human kidney transplants in 1963 and '64. Although one of the 13 patients lived for nine months, the rest died within a few weeks due to organ rejection or infections. Other surgeons attempted liver and heart xenotransplants, with similar results. Even the advent of the first immunosuppressant drug, cyclosporine, in 1983, did little to improve survival rates.
In the 1980s, Cooper—a pioneering heart transplant surgeon who'd embraced the dream of xenotransplantation—began arguing that apes and monkeys might not be the best donor animals after all. "First of all, there's not enough of them," he explains. "They breed in ones and twos, and take years to grow to full size. Even then, their hearts aren't big enough for a 70-kg. patient." Pigs, he suggested, would be a more practical alternative. But when he tried transplanting pig organs into nonhuman primates (as surrogates for human recipients), they were rejected within minutes.
In 1992, Cooper's team identified a sugar on the surface of porcine cells, called alpha-1,3-galactose (a-gal), as the main target for the immune system's attack. By then, the first genetically modified pigs had appeared, and biotech companies—led by the Swiss-based pharma giant Novartis—began pouring millions of dollars into developing one whose organs could elude or resist the human body's defenses.
Disaster struck five years later, when scientists reported that a virus whose genetic code was written into pig DNA could infect human cells in lab experiments. Although there was no evidence that the virus, known as PERV (for porcine endogenous retrovirus) could cause disease in people, the discovery stirred fears that xenotransplants might unleash a deadly epidemic. Facing scrutiny from government regulators and protests from anti-GMO and animal-rights activists, Novartis "pulled out completely," Cooper recalls. "They slaughtered all their pigs and closed down their research facility." Competitors soon followed suit.
The riddles surrounding animal-to-human transplants are far from fully solved.
A New Chapter – With New Questions
Yet xenotransplantation's visionaries labored on, aided by advances in genetic engineering and immunosuppression, as well as in the scientific understanding of rejection. In 2003, a team led by Cooper's longtime colleague David Sachs, at Harvard Medical School, developed a pig lacking the gene for a-gal; over the next few years, other scientists knocked out genes expressing two more problematic sugars. In 2013, Muhammad Mohiuddin, then chief of the transplantation section at the NHLBI, further modified a group of triple-knockout pigs, adding genes that code for two human proteins: one that shields cells from attack by an immune mechanism known as the complement system; another that prevents harmful coagulation. (It was those pigs whose hearts recently broke survival records when transplanted into baboon bellies. Mohiuddin has since become director of xenoheart transplantation at the University of Maryland's new Center for Cardiac Xenotransplantation Research.) And in August 2017, researchers at Harvard Medical School, led by George Church and Luhan Yang, announced that they'd used CRISPR-cas9—an ultra-efficient new gene-editing technique—to disable 62 PERV genes in fetal pig cells, from which they then created cloned embryos. Of the 37 piglets born from this experiment, none showed any trace of the virus.
Still, the riddles surrounding animal-to-human transplants are far from fully solved. One open question is what further genetic manipulations will be necessary to eliminate all rejection. "No one is so naïve as to think, 'Oh, we know all the genes—let's put them in and we are done,'" biologist Sean Stevens, another leading researcher, told the The New York Times. "It's an iterative process, and no one that I know can say whether we will do two, or five, or 100 iterations." Adding traits can be dangerous as well; pigs engineered to express multiple anticoagulation proteins, for example, often die of bleeding disorders. "We're still finding out how many you can do, and what levels are acceptable," says Cooper.
Another question is whether PERV really needs to be disabled. Cooper and some of his colleagues note that pig tissue has long been used for various purposes, such as artificial heart valves and wound-repair products, without incident; requiring the virus to be eliminated, they argue, will unnecessarily slow progress toward creating viable xenotransplant organs and the animals that can provide them. Others disagree. "You cannot do anything with pig organs if you do not remove them," insists bioethicist Jeantine Lunshof, who works with Church and Yang at Harvard. "The risk is simply too big."
"We've removed the cells, so we don't have to worry about latent viruses."
Meanwhile, over the past decade, other approaches to xenotransplantation have emerged. One is interspecies blastocyst complementation, which could produce organs genetically identical to the recipient's tissues. In this method, genes that produce a particular organ are knocked out in the donor animal's embryo. The embryo is then injected with pluripotent stem cells made from the tissue of the intended recipient. The stem cells move in to fill the void, creating a functioning organ. This technique has been used to create mouse pancreases in rats, which were then successfully transplanted into mice. But the human-pig "chimeras" recently created by scientists were destroyed after 28 days, and no one plans to bring such an embryo to term anytime soon. "The problem is that cells don't stay put; they move around," explains Father Kevin FitzGerald, a bioethicist at Georgetown University. "If human cells wind up in a pig's brain, that leads to a really interesting conundrum. What if it's self-aware? Are you going to kill it?"
Much further along, and less ethically fraught, is a technique in which decellularized pig organs act as a scaffold for human cells. A Minnesota-based company called Miromatrix Medical is working with Mayo Clinic researchers to develop this method. First, a mild detergent is pumped through the organ, washing away all cellular material. The remaining structure, composed mainly of collagen, is placed in a bioreactor, where it's seeded with human cells. In theory, each type of cell that normally populates the organ will migrate to its proper place (a process that naturally occurs during fetal development, though it remains poorly understood). One potential advantage of this system is that it doesn't require genetically modified pigs; nor will the animals have to be raised under controlled conditions to avoid exposure to transmissible pathogens. Instead, the organs can be collected from ordinary slaughterhouses.
Recellularized livers in bioreactors
(Courtesy of Miromatrix)
"We've removed the cells, so we don't have to worry about latent viruses," explains CEO Jeff Ross, who describes his future product as a bioengineered human organ rather than a xeno-organ. That makes PERV a nonissue. To shorten the pathway to approval by the Food and Drug Administration, the replacement cells will initially come from human organs not suitable for transplant. But eventually, they'll be taken from the recipient (as in blastocyst complementation), which should eliminate the need for immunosuppression.
Clinical trials in xenotransplantation may begin as early as 2020.
Miromatrix plans to offer livers first, followed by kidneys, hearts, and eventually lungs and pancreases. The company recently succeeded in seeding several decellularized pig livers with human and porcine endothelial cells, which flocked obediently to the blood vessels. Transplanted into young pigs, the organs showed unimpaired circulation, with no sign of clotting. The next step is to feed all four liver cell types back into decellularized livers, and see if the transplanted organs will keep recipient pigs alive.
Ross hopes to launch clinical trials by 2020, and several other groups (including Cooper's, which plans to start with kidneys) envision a similar timeline. Investors seem to share their confidence. The biggest backer of xenotransplantation efforts is United Therapeutics, whose founder and co-CEO, Martine Rothblatt, has a daughter with a lung condition that may someday require a transplant; since 2011, the biotech firm has poured at least $100 million into companies pursuing such technologies, while supporting research by Cooper, Mohiuddin, and other leaders in the field. Church and Yang, at Harvard, have formed their own company, eGenesis, bringing in a reported $40 million in funding; Miromatrix has raised a comparable amount.
It's impossible to predict who will win the xenotransplantation race, or whether some new obstacle will stop the competition in its tracks. But Jennifer Cisneros is rooting for all the contestants. "These technologies could save my life," she says. If she hasn't found another kidney before trials begin, she has just one request: "Sign me up."
Phages, which are harmless viruses that destroy specific bacteria, are becoming useful tools to protect our food supply.
Food producers and packagers fight bacteria by potent chemicals, with chlorine being the go-to disinfectant. Cattle carcasses, for example, are typically washed by chlorine solutions as the animals' intestines are removed. Leafy greens are bathed in water with added chlorine solutions. However, because the same "bath" can be used for multiple veggie batches and chlorine evaporates over time, the later rounds may not kill all of the bacteria, sparing some. The natural and organic producers avoid chlorine, substituting it with lactic acid, a more holistic sanitizer, but even with all these efforts, some pathogens survive, sickening consumers and causing food recalls. As we farm more animals and grow more produce, while also striving to use fewer chemicals and more organic growing methods, it will be harder to control bacteria's spread.
"It took us a long time to convince the FDA phages were safe and efficient alternatives. But we had worked with them to gather all the data they needed, and the FDA was very supportive in the end."
Luckily, bacteria have their own killers. Called bacteriophages, or phages for short, they are viruses that prey on bacteria only. Under the electron microscope, they look like fantasy spaceships, with oblong bodies, spider-like legs and long tails. Much smaller than a bacterium, phages pierce the microbes' cells with their tails, sneak in and begin multiplying inside, eventually bursting the microbes open—and then proceed to infect more of them.
The best part is that these phages are harmless to humans. Moreover, recent research finds that millions of phages dwell on us and in us—in our nose, throat, skin and gut, protecting us from bacterial infections as part of our healthy microbiome. A recent study suggested that we absorb about 30 billion phages into our bodies on a daily basis. Now, ingeniously, they are starting to be deployed as anti-microbial agents in the food industry.
A Maryland-based phage research company called Intralytix is doing just that. Founded by Alexander Sulakvelidze, a microbiologist and epidemiologist who came to the United States from Tbilisi, the capital of Georgia, Intralytix makes and sells five different FDA-approved phage cocktails that work against some of the most notorious food pathogens: ListShield for Listeria, SalmoFresh for Salmonella, ShigaShield for Shigella, another foodborne bug, and EcoShield for E.coli, including the infamous strain that caused the Jack in the Box outbreak in 1993 that killed four children and sickened 732 people across four states. Last year, the FDA granted its approval to yet another Intralytix phage for managing Campylobacter contamination, named CampyShield. "We call it safety by nature," Sulakvelidze says.
Intralytix grows phages inside massive 1500-liter fermenters, feeding them bacterial "fodder."
Photo credit: Living Radiant Photography
Phage preparations are relatively straightforward to make. In nature, phages thrive in any body of water where bacteria live too, including rivers, lakes and bays. "I can dip a bucket into the Chesapeake Bay, and it will be full of all kinds of phages," Sulakvelidze says. "Sewage is another great place to look for specific phages of interest, because it's teeming with all sorts of bacteria—and therefore the viruses that prey on them."
In lab settings, Intralytix grows phages inside massive 1500-liter fermenters, feeding them bacterial "fodder." Once phages multiply enough, they are harvested, dispensed into containers and shipped to food producers who have adopted this disinfecting practice into their preparation process. Typically, it's done by computer-controlled sprayer systems that disperse mist-like phage preparations onto the food.
Unlike chemicals like chlorine or antibiotics, which kill a wide spectrum of bacteria, phages are more specialized, each feeding on specific microbial species. A phage that targets salmonella will not prey on listeria and vice versa. So food producers may sometimes use a combo of different phage preparations. Intralytix is continuously researching and testing new phages. With a contract from the National Institutes of Health, Intralytix is expanding its automated high-throughput robot that tests which phages work best against which bacteria, speeding up the development of the new phage cocktails.
Phages have other "talents." In her recent study, Jaroni found that phages have the ability to destroy bacterial biofilms—colonies of microorganisms that tend to grow on surfaces of the food processing equipment, surrounding themselves with protective coating that even very harsh chemicals can't crack.
"Phages are very clever," Jaroni says. "They produce enzymes that target the biofilms, and once they break through, they can reach the bacteria."
Convincing the FDA that phages were safe to use on food products was no easy feat, Sulakvelidze says. In his home country of Georgia, phages have been used as antimicrobial remedies for over a century, but the FDA was leery of using viruses as food safety agents. "It took us a long time to convince the FDA phages were safe and efficient alternatives," Sulakvelidze says. "But we had worked with them to gather all the data they needed, and the FDA was very supportive in the end."
The agency had granted Intralytix its first approval in 2006, and over the past 10 years, the company's sales increased by over 15-fold. "We currently sell to about 40 companies and are in discussions with several other large food producers," Sulakvelidze says. One indicator that the industry now understands and appreciates the science of phages was that his company was ranked as Top Food Safety Provider in 2021 by Food and Beverage Technology Review, he adds. Notably, phage sprays are kosher, halal and organic-certified.
Intralytix's phage cocktails to safeguard food from bacteria are approved for consumers in addition to food producers, but currently the company sells to food producers only. Selling retail requires different packaging like easy-to-use spray bottles and different marketing that would inform people about phages' antimicrobial qualities. But ultimately, giving people the ability to remove pathogens from their food with probiotic phage sprays is the goal, Sulakvelidze says.
It's not the company's only goal. Now Intralytix is going a step further, investigating phages' probiotic and therapeutic abilities. Because phages are highly specialized in the bacteria they target, they can be used to treat infections caused by specific pathogens while leaving the beneficial species of our microbiome intact. In an ongoing clinical trial with Mount Sinai, Intralytix is now investigating a potential phage treatment against a certain type of E. coli for patients with Crohn's disease, and is about to start another clinical trial for treating bacterial dysentery.
"Now that we have proved that phages are safe and effective against foodborne bacteria," Sulakvelidze says, "we are going to demonstrate their potential in therapeutic applications."
This article was first published by Leaps.org on October 27, 2021.