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."
Today's podcast guest, Dr. Renee Wegrzyn, directs ARPA-H, a new agency formed last year to spearhead health innovations. Time will tell if ARPA-H will produce advances on the level of its fellow agency, DARPA.
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How will the agency figure out which projects to take on, especially with so many patient advocates for different diseases demanding moonshot funding for rapid progress?
I talked with Dr. Wegrzyn about the opportunities and challenges, what lessons ARPA-H is borrowing from Operation Warp Speed, how she decided on the first ARPA-H project that was announced recently, why a separate agency was needed instead of reforming HHS and the National Institutes of Health to be better at innovation, and how ARPA-H will make progress on disease prevention in addition to treatments for cancer, Alzheimer’s and diabetes, among many other health priorities.
Dr. Wegrzyn’s resume leaves no doubt of her suitability for this role. She was a program manager at DARPA where she focused on applying gene editing and synthetic biology to the goal of improving biosecurity. For her work there, she received the Superior Public Service Medal and, in case that wasn’t enough ARPA experience, she also worked at another ARPA that leads advanced projects in intelligence, called I-ARPA. Before that, she ran technical teams in the private sector working on gene therapies and disease diagnostics, among other areas. She has been a vice president of business development at Gingko Bioworks and headed innovation at Concentric by Gingko. Her training and education includes a PhD and undergraduate degree in applied biology from the Georgia Institute of Technology and she did her postdoc as an Alexander von Humboldt Fellow in Heidelberg, Germany.
Dr. Wegrzyn told me that she’s “in the hot seat.” The pressure is on for ARPA-H especially after the need and potential for health innovation was spot lit by the pandemic and the unprecedented speed of vaccine development. We'll soon find out if ARPA-H can produce gamechangers in health that are equivalent to DARPA’s creation of the internet.
Show links:
ARPA-H - https://arpa-h.gov/
Dr. Wegrzyn profile - https://arpa-h.gov/people/renee-wegrzyn/
Dr. Wegrzyn Twitter - https://twitter.com/rwegrzyn?lang=en
President Biden Announces Dr. Wegrzyn's appointment - https://www.whitehouse.gov/briefing-room/statement...
Leaps.org coverage of ARPA-H - https://leaps.org/arpa/
ARPA-H program for joints to heal themselves - https://arpa-h.gov/news/nitro/ -
ARPA-H virtual talent search - https://arpa-h.gov/news/aco-talent-search/
Dr. Renee Wegrzyn was appointed director of ARPA-H last October.
Tardigrades can completely dehydrate and later rehydrate themselves, a survival trick that scientists are harnessing to preserve medicines in hot temperatures.
Formulating biologics to withstand drying and hot temperatures has been the holy grail for pharmaceutical researchers for decades. It’s a hard feat to manage. “Biologic pharmaceuticals are highly efficacious, but many are inherently unstable,” says Thomas Boothby, assistant professor of molecular biology at University of Wyoming. Therefore, during storage and shipping, they must be refrigerated at 2 to 8 degrees Celsius (35 to 46 degrees Fahrenheit). Some must be frozen, typically at -20 degrees Celsius, but sometimes as low -90 degrees Celsius as was the case with the Pfizer Covid vaccine.
For Covid, fewer than 73 percent of the global population received even one dose. The need for refrigerated or frozen handling was partially to blame.
The costly cold chain
The logistics network that ensures those temperature requirements are met from production to administration is called the cold chain. This cold chain network is often unreliable or entirely lacking in remote, rural areas in developing nations that have malfunctioning electrical grids. “Almost all routine vaccines require a cold chain,” says Christopher Fox, senior vice president of formulations at the Access to Advanced Health Institute. But when the power goes out, so does refrigeration, putting refrigerated or frozen medical products at risk. Consequently, the mRNA vaccines developed for Covid-19 and other conditions, as well as more traditional vaccines for cholera, tetanus and other diseases, often can’t be delivered to the most remote parts of the world.
To understand the scope of the challenge, consider this: In the U.S., more than 984 million doses of Covid-19 vaccine have been distributed so far. Each one needed refrigeration that, even in the U.S., proved challenging. Now extrapolate to all vaccines and the entire world. For Covid, fewer than 73 percent of the global population received even one dose. The need for refrigerated or frozen handling was partially to blame.
Globally, the cold chain packaging market is valued at over $15 billion and is expected to exceed $60 billion by 2033.
Adobe Stock
Freeze-drying, also called lyophilization, which is common for many vaccines, isn’t always an option. Many freeze-dried vaccines still need refrigeration, and even medicines approved for storage at ambient temperatures break down in the heat of sub-Saharan Africa. “Even in a freeze-dried state, biologics often will undergo partial rehydration and dehydration, which can be extremely damaging,” Boothby explains.
The cold chain is also very expensive to maintain. The global pharmaceutical cold chain packaging market is valued at more than $15 billion, and is expected to exceed $60 billion by 2033, according to a report by Future Market Insights. This cost is only expected to grow. According to the consulting company Accenture, the number of medicines that require the cold chain are expected to grow by 48 percent, compared to only 21 percent for non-cold-chain therapies.
Tardigrades to the rescue
Tardigrades are only about a millimeter long – with four legs and claws, and they lumber around like bears, thus their nickname – but could provide a big solution. “Tardigrades are unique in the animal kingdom, in that they’re able to survive a vast array of environmental insults,” says Boothby, the Wyoming professor. “They can be dried out, frozen, heated past the boiling point of water and irradiated at levels that are thousands of times more than you or I could survive.” So, his team is gradually unlocking tardigrades’ survival secrets and applying them to biologic pharmaceuticals to make them withstand both extreme heat and desiccation without losing efficacy.
Boothby’s team is focusing on blood clotting factor VIII, which, as the name implies, causes blood to clot. Currently, Boothby is concentrating on the so-called cytoplasmic abundant heat soluble (CAHS) protein family, which is found only in tardigrades, protecting them when they dry out. “We showed we can desiccate a biologic (blood clotting factor VIII, a key clotting component) in the presence of tardigrade proteins,” he says—without losing any of its effectiveness.
The researchers mixed the tardigrade protein with the blood clotting factor and then dried and rehydrated that substance six times without damaging the latter. This suggests that biologics protected with tardigrade proteins can withstand real-world fluctuations in humidity.
Furthermore, Boothby’s team found that when the blood clotting factor was dried and stabilized with tardigrade proteins, it retained its efficacy at temperatures as high as 95 degrees Celsius. That’s over 200 degrees Fahrenheit, much hotter than the 58 degrees Celsius that the World Meteorological Organization lists as the hottest recorded air temperature on earth. In contrast, without the protein, the blood clotting factor degraded significantly. The team published their findings in the journal Nature in March.
Although tardigrades rarely live more than 2.5 years, they have survived in a desiccated state for up to two decades, according to Animal Diversity Web. This suggests that tardigrades’ CAHS protein can protect biologic pharmaceuticals nearly indefinitely without refrigeration or freezing, which makes it significantly easier to deliver them in locations where refrigeration is unreliable or doesn’t exist.
The tricks of the tardigrades
Besides the CAHS proteins, tardigrades rely on a type of sugar called trehalose and some other protectants. So, rather than drying up, their cells solidify into rigid, glass-like structures. As that happens, viscosity between cells increases, thereby slowing their biological functions so much that they all but stop.
Now Boothby is combining CAHS D, one of the proteins in the CAHS family, with trehalose. He found that CAHS D and trehalose each protected proteins through repeated drying and rehydrating cycles. They also work synergistically, which means that together they might stabilize biologics under a variety of dry storage conditions.
“We’re finding the protective effect is not just additive but actually is synergistic,” he says. “We’re keen to see if something like that also holds true with different protein combinations.” If so, combinations could possibly protect against a variety of conditions.
Commercialization outlook
Before any stabilization technology for biologics can be commercialized, it first must be approved by the appropriate regulators. In the U.S., that’s the U.S. Food and Drug Administration. Developing a new formulation would require clinical testing and vast numbers of participants. So existing vaccines and biologics likely won’t be re-formulated for dry storage. “Many were developed decades ago,” says Fox. “They‘re not going to be reformulated into thermo-stable vaccines overnight,” if ever, he predicts.
Extending stability outside the cold chain, even for a few days, can have profound health, environmental and economic benefits.
Instead, this technology is most likely to be used for the new products and formulations that are just being created. New and improved vaccines will be the first to benefit. Good candidates include the plethora of mRNA vaccines, as well as biologic pharmaceuticals for neglected diseases that affect parts of the world where reliable cold chain is difficult to maintain, Boothby says. Some examples include new, more effective vaccines for malaria and for pathogenic Escherichia coli, which causes diarrhea.
Tallying up the benefits
Extending stability outside the cold chain, even for a few days, can have profound health, environmental and economic benefits. For instance, MenAfriVac, a meningitis vaccine (without tardigrade proteins) developed for sub-Saharan Africa, can be stored at up to 40 degrees Celsius for four days before administration. “If you have a few days where you don’t need to maintain the cold chain, it’s easier to transport vaccines to remote areas,” Fox says, where refrigeration does not exist or is not reliable.
Better health is an obvious benefit. MenAfriVac reduced suspected meningitis cases by 57 percent in the overall population and more than 99 percent among vaccinated individuals.
Lower healthcare costs are another benefit. One study done in Togo found that the cold chain-related costs increased the per dose vaccine price up to 11-fold. The ability to ship the vaccines using the usual cold chain, but transporting them at ambient temperatures for the final few days cut the cost in half.
There are environmental benefits, too, such as reducing fuel consumption and greenhouse gas emissions. Cold chain transports consume 20 percent more fuel than non-cold chain shipping, due to refrigeration equipment, according to the International Trade Administration.
A study by researchers at Johns Hopkins University compared the greenhouse gas emissions of the new, oral Vaxart COVID-19 vaccine (which doesn’t require refrigeration) with four intramuscular vaccines (which require refrigeration or freezing). While the Vaxart vaccine is still in clinical trials, the study found that “up to 82.25 million kilograms of CO2 could be averted by using oral vaccines in the U.S. alone.” That is akin to taking 17,700 vehicles out of service for one year.
Although tardigrades’ protective proteins won’t be a component of biologic pharmaceutics for several years, scientists are proving that this approach is viable. They are hopeful that a day will come when vaccines and biologics can be delivered anywhere in the world without needing refrigerators or freezers en route.