Meet the exosome, the rising star in drug delivery

Millions of tiny bubbles, released from cells and packaged with molecular mail, are racing through your bloodstream right now. And until recently, only a handful of researchers gave them any thought.

Stephen J. Gould is one of those scientists. For more than a decade, Gould has devoted significant time and resources to understanding the curious cellular couriers. Called exosomes, these lipid vesicles shuttle proteins and genetic information between both neighboring and distant cells. “They are just a ubiquitous fact of our biology,” the Johns Hopkins University School of Medicine professor says.

Now they’re positioned to become a widespread tool for drug delivery.

Scientists have known about exosomes for decades, but as recently as 2006, only 508, mostly obscure, papers referred to them, according to PubMed. Today, a search on the site brings more than 8,000 hits, including several high-profile publications from the past year.

That research explosion is due, in part, to Swedish scientist Jan Lötvall from the University of Gothenburg. Exosomes had long been viewed as merely tiny trash sacs tossed from cells, but Lötvall showed in 2007 that some cells use exosomes to transfer genetic material—messenger RNAs to make proteins and microRNAs to regulate the expression of genes—between each other (Nat. Cell Biol. 2007, DOI: 10.1038/ncb1596). That discovery set scientists searching for ways that exosomes might be involved in health and disease and even be used as treatments.

“There was huge skepticism at first, and there is still some out there,” Lötvall says.

Now it is clear that Lötvall’s study wasn’t a fluke. The vesicles are implicated in spreading diseases, including cancer, and metabolic conditions, like diabetes and obesity. A recent study even points to exosomes as a culprit for distributing amyloid-β, the plaque-forming protein that accumulates in the brains of people with Alzheimer’s disease. If exosomes can so easily carry molecules that spread disease, scientists began thinking they might be useful to carry molecules that stop disease.

“Lötvall changed the way people think about exosomes,” says Doug Williams, CEO of Codiak BioSciences. “It’s become clear that this is a very important and ancient messenger system.”

Codiak is one of a growing number of biotech start-ups attempting to hijack that messenger system to ferry drugs into cells in parts of the body, like the brain, that would otherwise be difficult to reach. Codiak is an investor darling that has raised nearly $170 million since 2016, but more than a dozen other companies have also begun working on exosome-based therapies in the past few years. Some are backed by peer-reviewed research; others are dubiously peddling exosomes as a cure-all.

Like Codiak, many are manipulating exosomes to solve drug delivery problems for a dizzying array of therapies: small molecules, RNA therapies, proteins, viral gene therapy, and even CRISPR gene-editing tools. Studies in cells and animals suggest that exosomes could be the gift wrapping for them all.

Still others are capitalizing on research that suggests the exosomes themselves, when derived from stem cells, could become a new branch of regenerative medicine. Several firms have already convinced big pharma companies to use their technology.

To Gould, the sudden spurt of interest is shocking. Just two years ago, “people would say we are pretty close to understanding the biology but a ways off from the therapeutics and diagnostics,” he says. Yet already, exosome-based cancer diagnostics are available, and multiple exosome therapies could begin testing in clinical trials next year.

Drug distributors

Williams remembers first chatting about exosomes with his Biogen colleagues soon after he joined the drug company as vice president of R&D in 2011. A study from Matthew J. A. Wood’s group at the University of Oxford was making the rounds for its shocking demonstration that exosomes stuffed full of small interfering RNA (siRNA) could reach cells inside the brains of mice (Nat. Biotechnol. 2011, DOI: 10.1038/nbt.1807). Once past the brain’s protective barrier, the genetic material lowered production of BACE1, a protein involved in Alzheimer’s disease. “I was really intrigued by the notion, but we never actually did anything with it at Biogen,” Williams says.

Then in 2015, during a visit to the University of Texas MD Anderson Cancer Center, Williams learned about work going on in the labs of cancer biologists Raghu Kalluri and Valerie LeBleu. They were using exosomes to successfully deliver an siRNA therapy to cancer cells in mice.

The research had the potential to overcome a problem scientists have wrestled with for over a decade: getting siRNA into the right cells. Synthetic lipid nanoparticles have been the mainstay delivery device for siRNA and other RNA therapies, but they can cause a toxic immune response in humans. And although their design has improved over the years, they remain notorious for congregating in the liver, limiting the kinds of diseases drugmakers are able to effectively target.

Kalluri’s data showed that although many exosomes also gather in the liver, significant numbers of them manage to travel to other organs, including the pancreas. His team used them to deliver siRNA that blocked production of a mutant protein called KRas, one of the most “undruggable” cancer targets.

Intravenous injections of Kalluri’s siRNA-loaded exosomes suppressed pancreatic cancer in mice better than similar injections of siRNA-loaded lipid nanoparticles, and without any obvious immune reactions (Nature 2017, DOI: 10.1038/nature22341). “That was the aha moment for me,” Williams recalls.

Kalluri linked up with Eric Lander of Broad Institute of MIT & Harvard to cofound Codiak, and Williams left Biogen to become the start-up’s CEO. Codiak is now advancing its own KRas program alongside others that use exosomes to deliver small molecules and peptides. “It seems like all cells make exosomes, and all cells can take them up,” Williams says. “Exosomes are amenable to every therapeutic area.”

MD Anderson’s LeBleu hopes that the cancer center will start a clinical trial this year or next testing her and Kalluri’s pancreatic cancer therapy. Their labs have siRNA-loaded exosomes tackling other targets as well. “We may have landed on a tool that could revolutionize the undruggable approach,” she says.

Other companies are sprouting up too. “The field is expanding massively,” says Antonin de Fougerolles, CEO of Evox Therapeutics, a company cofounded by Wood, the Oxford professor who used exosomes to get siRNA into mice brains. Evox is developing exosome-based treatments for rare diseases, particularly conditions that require delivery into the brain. The start-up has formed partnerships with the German giant Boehringer Ingelheim and a second, unnamed drug company.

Another start-up, Anjarium Biosciences, is creating so-called hybridosomes, a mash-up of synthetic lipid nanoparticles and natural exosomes. A group led by Casey Maguire at Massachusetts General Hospital has packaged an adeno-associated virus gene therapy inside an exosome—he calls the combo a vexosome—to transport the virus into cells it would normally have a hard time reaching, such as sensory cells in the inner ear (Mol. Ther. 2016, DOI: 10.1016/j.ymthe.2016.12.010). And another team recently used microvesicles—a larger cousin to exosomes—to deliver proteins, mRNA, and the CRISPR/Cas9 gene-editing system into cells (Nat. Commun. 2018, DOI: 10.1038/s41467-018-03390-x).

Engineering aside, if exosomes prove to be efficacious drug delivery vehicles, their success will likely arise from their compatibility with the human body. That’s why several groups are looking for alternative sources of natural exosomes. Others think that exosomes on their own will treat a number of diseases.

Exosome powers

Stella Kourembanas and S. Alex Mitsialis didn’t plan on becoming exosome experts. The two Boston Children’s Hospital scientists have spent more than a decade trying to tap the potential of stem cells to create regenerative therapies for newborn babies with severely damaged lungs. Their team demonstrated that human mesenchymal stem cells (MSCs)—adult stem cells that can turn into many kinds of cells—can treat animal models of lung disease by reducing inflammation and repairing lung tissue.

Their first guess was that the MSCs work by melding with the damaged tissue and becoming lung cells. But a closer look revealed that the donor stem cells didn’t stick around. “It was a paradox,” Mitsialis says.

That led the team on a years-long quest to explain the cells’ regenerative properties. In 2012, the researchers finally showed that extracellular vesicles released from MSCs were preventing lung damage in mice. The group then spent several years learning how to isolate and purify exosomes from stem cells. Last year, they showed that MSC-derived exosomes are responsible for the healing effect in mice with a serious lung disease (Am. J. Respir. Crit. Care Med. 2017, DOI: 10.1164/rccm.201705-0925oc).

Now the team is in talks with the U.S. Food & Drug Administration and an undisclosed industry partner to use exosomes to treat that same disease, called bronchopulmonary dysplasia, in newborn babies, where the prevalence is rising in step with the increasing survival rates of preterm infants.

“The FDA wants to make sure we treat the sickest babies with the highest risk,” says Kourembanas, who is also chief of the newborn medicine division at Boston Children’s. “I think in rushing to get stem cells into the clinic there were a lot of steps not done carefully,” she says. “We can’t repeat that with exosomes.”

In many cases, it is not clear what molecules inside the exosomes have therapeutic properties, but several stem cell companies are pivoting to exosomes nonetheless. For example, Capricor Therapeutics, which was founded in 2005 to develop stem cell therapies derived from heart tissue, is now using exosomes derived from those same cells to try to treat cardiac and inflammatory conditions. Capricor has a partnership with the U.S. Army to treat trauma-related conditions in the field with exosomes.

ArunA Biomedical, which was founded in 2003, recently tested exosomes derived from human MSCs and neural stem cells in rodent and pig models of stroke. The firm reported improved preservation of brain cells and motor movement in the animals (Stroke 2018, DOI: 10.1161/strokeaha.117.020353). Clinical trials with the exosomes could begin in humans by 2020, ArunA CEO Steven Stice says.

Other companies are taking a less cautious approach, forgoing traditional clinical trials and FDA approval and heading straight to consumers. Typically rogue stem cell therapy clinics, these firms are tempting customers with unproven claims suggesting that exosomes can aid in aesthetics, erectile dysfunction, immunotherapy, orthopedics, and more.

While stem cells have performed wonders in animal studies, success in humans has been harder to find. Even in animal studies, it is unclear what makes stem-cell-derived exosomes therapeutic. “This is the question from every single presentation that I give,” says Gareth Willis, a researcher on the Boston Children’s Hospital team. He says to “think of the exosome not as an instrument but as an orchestra.” The menagerie of proteins and RNA molecules is likely more important than any single compound.

Some scientists, including stem cell biologist Raj Kishore, hope to elucidate that molecular mystery. Kishore and his colleagues at Temple University won an $11.6 million grant from the National Institutes of Health last year to study stem-cell-derived exosomes in heart repair.

“We should try to understand the mechanism a little more and not rush the therapies,” Kishore says. “I don’t want to see this field die like the stem cell therapy field, which took a setback from not understanding how the mechanisms work.”

Even as academic labs and biotech firms blaze ahead with exosomes to deliver therapeutics or use the vesicles as therapeutics themselves, they are confronted with a bevy of technical issues. “Just making enough of the vesicles is one of the big challenges,” says Xandra O. Breakefield, a researcher at Massachusetts General Hospital. Right now scientists collect exosomes that are spit out from cells grown in the lab. “Then once you get your vesicles, there is a lot of disagreement about how to isolate them.”

Another problem is extracellular vesicle diversity. Whereas researchers once recognized just two major classes, exosomes and microvesicles, “now we realize there are many other types of vesicles out there, and we don’t know how to separate them,” Breakefield says.

“It complicates a lot of people’s experiments,” adds Johns Hopkins’s Gould, who is also a scientific adviser to Tavec Pharmaceuticals, a start-up fundraising to develop anticancer exosomes. Consistency and reproducibility in experiments with exosome-based therapies could be compromised if all the exosomes are different sizes and thus loaded with different amounts of drugs.

As some scientists grapple with the wide diversity of exosomes produced from a single cell line, others are seeking diversity. One start-up is hunting for new kinds of exosomes from bacteria, fungi, plants, and animals. Another group is developing fruit-derived exosome therapies to deliver cancer drugs.

The pharmaceutical industry is starting to pay attention too. Earlier this month, Roche agreed to pay PureTech Health up to $36 million for access to its exosomes extracted from dairy cow milk. “Mammalian milk is loaded with exosomes,” says Joseph Bolen, PureTech’s chief scientific officer.

Roche intends to package antisense oligonucleotide therapies in the exosomes for oral delivery. Bolen says the same trick might work for other nucleic acid and biologic therapies, which are typically injected. “We absolutely believe this will have widespread application,” he says.

Almost everyone working on exosome therapies is tackling a different disease, with different exosomes. And they’re doing it fast. That could help catapult the field forward or make it difficult to troubleshoot as problems arise. Breakefield sees shadows from the early days of gene therapy in the exosome field.

“It is a little bit of a dangerous time for this field,” she says. “Because if we get some happy-go-lucky guy that is so confident it will work, and things go wrong, it will set everybody back.”

Scientists who weathered years of skepticism are optimistic, though. “Almost nobody got into this field because they wanted to,” Gould says. Instead, he sees the migration toward exosomes as the result of scientists chasing down the basic biology of a disease, developing a diagnostic, or trying to find a better way to deliver drugs.

“It speaks to the fact that they are important in almost every aspect of biology and medicine,” Gould says. “The potential here is huge.”