Monthly Archives: April 2011

Exosomes: The Little Vesicles That Could

A Boston Biotech Watch guest post by Dima Ter-Ovanesyan*

When I mention to other biologists that I work on exosomes, I am used to getting blank stares. Exosomes, also sometimes called microvesicles, are small lipid vesicles secreted by all cells. Like many dark corners of biology, the exosome field is a province of a few experts and is still largely unknown to the mainstream. But thanks to some exciting early results and a long list of potential medical applications, exosomes are beginning to move out of the shadows and into the light. Exosomes were the subject of two sessions at the annual American Association of Cancer Research (AACR)  meeting earlier this month. In January, the first-ever conference on exosomes brought together more than two hundred researchers from around the world. And now even the popular press is picking up on the applications of exosomes to RNA interference (RNAi) drug delivery and diagnostics in fields ranging from cancer to diabetes.

The concept of cells budding off small particles was actually first mentioned in Charles Darwin’s The Variation of Plants and Animals Under Domestication in 1868 and exosomes were first observed by electron microscopy in the 1980s. For twenty years after that, exosomes were thought by many to be nothing more than “cellular trash bags” that dump proteins deemed to have outlived their “use-by” date. This changed a few years ago, though, when a number of independent studies showed that exosomes actually contain not only protein but also RNA. This discovery opened the possibility of using the RNA in exosomes floating around in bodily fluids to learn all kinds of secrets about the cells that release them.

Charles Darwin

There first (as usual)

I learned about the diagnostic potential of exosomes in early 2009 when a venture capitalist asked my opinion on a business plan. The company, called Exosome Diagnostics, was being spun out of Massachusetts General Hospital (MGH) to develop new diagnostics based on analyzing the RNA in exosomes isolated from blood and urine. At the time, I was an undergraduate at MIT working on microRNAs. After reading the business plan, the patent filing, and the underlying scientific publication, I called the VC back. “This could be HUGE,” I told him. “We should get in on this,” he replied, and promptly forgot all about it. I did not. After graduation, I opted to learn more about exosomes through a brief stint at the Curie Institute in Paris. I then joined Exosome Diagnostics, which by then was flush with a $20M Series A.

The company’s platform is based on the work done at MGH, which showed that mutant RNA transcripts derived from key genes can be detected in exosomes released by cancer cells. Johan Skog and colleagues had shown that by isolating exosomes from blood and looking at the RNA inside, they could tell whether a patient’s tumor contained mutant EGF Receptor (EGFR) – establishing the proof of principle for exosomes as companion diagnostics. Oncologists could use them to peer into the genetics of tumor and decide whether a patient would be a good candidate for EGFR-inhibiting drugs such as Tarceva® and Iressa®. As pharma companies move increasingly towards targeted cancer therapies coupled to companion diagnostics (think PLX-4032, Plexxikon’s BRAF inhibitor for melanoma, or Crizotinib, Pfizer’s ALK inhibitor for lung cancer), the appeal of a blood or urine test instead of a biopsy to analyze specific mutations is not hard to see. This is particularly advantageous in cancers where obtaining a biopsy is difficult, like brain cancer or lung cancer.

I think that companion diagnostics in cancer are just the tip of the iceberg for exosomes in diagnostics. Several recent studies have shown that different levels of certain RNAs in exosomes and other vesicles isolated from blood correlate with different disease states. In other words, exosomes have RNA “signatures” for different diseases. One recently published study , for example, showed that a specific microRNA in vesicles derived from diabetics’ blood was elevated compared to vesicles taken from non-diabetics. Amazingly, the researchers also showed that this biomarker could potentially be used to identify patients who will get diabetes before any clinical symptoms occur. Although the test in the paper was not yet sensitive enough for the clinic, the results raise the intriguing possibility of using RNA signatures to predict disease, not just diagnose it.

Several reports at the January exosome conference highlighted diagnostic applications of profiling exosomal RNA in different diseases:

  1. Signatures of mRNA isolated from exosomes in the blood could be used to classify brain tumors based on aggressiveness.
  2. A specific microRNA isolated from exosomes in the cerebrospinal fluid (CSF) was found in brain trauma patients but not in healthy controls.
  3. Certain microRNAs isolated from exosomes in the blood of pregnant women could be used to predict premature births.

Although larger studies will be needed to confirm these effects, I imagine that, in the not too distant future, exosomes will join the list of proteins and metabolites currently profiled during routine blood draws. Through methods such as high-throughput sequencing, the RNA inside exosomes (dare I say “exo-transcriptome”?) will be analyzed and will prove spectacularly useful in helping physicians assess and track patient health.

Exosome Micrograph

I spy a novel class of biomarkers (Image courtesy Johan Skog and Casey Maguire, Massachusetts General Hospital)

Exciting as the potential of exosomes is in diagnostics, you may be wondering what they actually do. Are the different RNA profiles in exosomes more than a correlation? Do exosomes actually contribute to disease? This was an intense topic of debate at the exosome conference, and, after listening to four days of presentations on the topic, I would venture to say that we still really don’t know. The fact that exosomes contain RNA raises the extremely intriguing possibility that cells throughout the body communicate by sending each other little “packages” of RNA, and the RNA can then perhaps act in the recipient cells – be translated into protein in the case of mRNA or repress the expression of other genes in the case of microRNAs. And, if this is the case, one could imagine hijacking this pathway to deliver therapeutic RNAs and thereby overcome an enormous roadblock.

The main obstacle to realizing the tremendous potential of RNAi therapeutics is the challenge of delivery. Whether or not exosomes are actually used to transfer RNA between cells remains to be proven, but results presented at the exosome conference and recently published by a group at Oxford made the first attempt at this approach. Led by Matthew Wood, the group loaded modified exosomes with an siRNA designed to knock down the BACE1 gene implicated in Alzheimer’s Disease.  Although they used healthy mice as models, the researchers demonstrated proof-of-principle by showing reduced levels of BACE1 in the brain. Of course, it would be reassuring to know what exosomes actually do in the body before injecting them into patients, especially given that exosome-based drugs would be complex biologics composed of several different proteins, not just lipid vesicles. As we learn more about the biology of exosomes, however, I could imagine scientists designing exosome-mimicking particles with the minimum necessary components to carry the RNA therapeutic to its designated site in the body.

As evidenced at the exosome sessions at the AACR meeting, there is increasing interest in exosomes secreted by cancer cells. Cancer exosomes have been found to contain oncogenic mRNA and proteins, and it is thus tempting to speculate that exosomes may have a role in modifying the tumor microenvironment and helping the cancer spread. If true, one could even imagine using therapeutics to specifically target cancer exosomes. Unlike with the use of exosomes for diagnostics, however, the use of exosomes for therapeutics will require scientists to uncover some of the fundamental biology of what exosomes are and how they function. Luckily, as evidenced by the excitement at the exosome conference, there are at least two hundred of them up for the challenge.

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*Dima Ter-Ovanesyan (dimatero@gmail.com) graduated from MIT in 2010 with a Bachelor of Science in Biology. At MIT, he worked on RNAi screens in cancer with Michael Hemann and microRNA targeting with Chris Burge and David Bartel. He was also an associate at CBT Advisors. He currently works at Exosome Diagnostics.

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Hacking Ourselves: “Biopunk: DIY Scientists Hack the Software of Life” by Marcus Wohlsen

April 14, 2011

A Boston Biotech Watch Book Review

By Steve Dickman, CEO, CBT Advisors

Marcus Wohlsen’s ahead-of-the-curve new book Biopunk: DIY Scientists Hack the Software of Life, brings us a radical idea: garage biologists are busily “hacking” their own genomes, cooking up a variety of novel and potentially useful wetware inventions. Some of these may look like Rube Goldberg contraptions right now, but they might change the world profoundly, much as mainstream biotechnology already has. Even (especially?) for those of us who live and breathe biotech in Cambridge, Massachusetts, this idea is fresh, even startling.

Steampunk personified

So much DNA, so little time*

Biopunk chronicles, for one, a young MIT-trained “DIY scientist” who created and ran a DNA test on herself in her Cambridge kitchen for less than $200. The test would cost thousands if ordered from a clinical lab. She used countertop gear to look for – and find – evidence that she had a predisposition for a hereditary and severe disease.

In another chapter, a research team in an undisclosed location crowdsources funds on the Internet to create “the world’s smallest version of the thermal cycler,” an all-important DNA analysis tool that would “wedge open the door … to peer-to-peer biotech.” Combined with “an as-yet hypothetical DNA reading chip” and some samples of pathogen DNA, the team’s invention could give a doctor or nurse working in the field in a developing country “an answer in minutes” about which pathogen had infected a patient.

And more: industrial-strength “DNA photocopiers” known as PCR machines encased not in sheet metal but in wood; an edgy conference called “Outlaw Biology?”; and a pony-tailed bioinformaticist who tinkers after hours in his Mountain View garage with a device that could read DNA electronically, a device that he would give away or sell at cost to developing-world health initiatives or to other biohackers. If it works, it could eventually undermine or augment traditional diagnostic assays based on technologies like ELISA and microarrays.

It is for the developing world, with its cost constraints, lack of up-to-date technology and urgent needs, for which biohacking would seem to hold the greatest promise, as long as it can overcome some daunting obstacles. But for would-be startup founders who need a shortcut to intellectual property, DIY would seem to offer an attractive “quick-and-dirty” alternative. No less a luminary than Freeman Dyson is a full-on advocate for DIY biology. In a 2007 essay entitled “Our Biotech Future” published in the New York Review of Books, he said “I predict that the domestication of biotechnology will dominate our lives during the next fifty years at least as much as the domestication of computers has dominated our lives during the previous fifty years.”

Marcus Wohlsen

Marcus Wohlsen

Wohlsen, a Bay Area-based science reporter for the Associated Press, plucked the book’s core concept from a brief story he had published. Unfortunately, the moment he has chosen to expand it into a book feels a bit too early. Invisible on Google Trends, “Biopunk” has been mentioned only in a few magazine articles (for example in the New Yorker and Wired), mostly in the context of the promise and threat of mass-producing DNA via “synthetic biology.”

But Wohlsen’s timing does society a favor. Although his choice of topic may not help his book ring the gong of popular science as did, say, James Gleick’s Chaos in the 1980s or Dava Sobel’s Longitude in the 1990s, he has nonetheless caught and illuminated biohacking while it is still a tiny subculture and yet potentially could grow into a powerful force. Could it become a bigger one? Could it – pardon the expression – go viral, with astonishing results? Or will it be tamed and shackled, reduced to a harmless hobby like coin collecting or trainspotting?

Wohlsen is a fine writer with an ear for the absurd. Biopunk is well written, well-organized and has a satisfying amount of fresh material, answering the insistent question “Who ARE these people?” in a way that brings the individuals satisfyingly to life.

But as enjoyable as the book is, it does not describe a “what is” as much as it gives us a glimpse of a “what might be.” Like personal computing before Steve Jobs and the Homebrew Computing Club, DIY DNA is missing both a galvanizing new technology (the personal computer, the internet) and a recognized leader.

YOUNG-STEVE-JOBS-APPLE-INC

Will the next Jobs hack bio?

What’s more, no matter how good it is, a non-fiction book cannot yet capture the world that may yet be created by DNA “hackers.” There are three reasons for this:

  • Reason Two: The possibilities are so massive no one can think of them yet. One could argue that financial or intellectual limitations will prevent the hacking of anything more complex than a bacterium. But that would be so wrong. Wohlsen shows convincingly that the technology of DNA manipulation is available, affordable and already being applied. So what if the “tinkerers,” as they proudly call themselves, have not yet tinkered their way to a gryphon or some other creature we have not even thought of yet? This technology – cloning, sequencing, DNA manipulation and now synthesis – is extremely powerful. Think about that power – to create and fuse entire genomes. Now consider the tools of the moment – of Facebook, Twitter, the internet itself — all tools that foster “distributed intelligence” and group problem-solving. Combine cheap tools to manipulate DNA with the power of networks and you’d better stand back because what happens might rival the power of the nuclear bomb, a comparison Wohlsen aptly draws.
Gryphon

Made to order?

  • Reason Three: Before they can create much, the government will stop them. This, it seems to me, is the biggest threat that faces the nascent and promising movement of DNA hacking. As Germans from both East and West Germany used to say just after reunification, the “wall in the head” is much more formidable and hard to dismantle than the actual Berlin Wall was. According to Wohlsen, the troubling arrest and successful prosecution of one apparently non-malicious DNA hacker has already shown that it can chill the field. More systematic government intervention has the potential to freeze it.

If government intervention – through regulation, post-9/11 bioterror laws or “just” by intimidation – were to shut down DNA hacking, this would be both sad and ironic. Sad because it would cut off a potentially limitless source of new discoveries that could benefit humankind. Ironic because forcing DNA hackers back into “societally approved” (read expensive, cumbersome, peer-reviewed) channels or driving them underground would be a denial of the bottom-up, can-do pioneering spirit that is part of the cultural heritage of the United States.

The not so subtle message of Wohlsen’s book is that the very nature of the hacker community – low-budget, decentralized and interested in the pursuit of novel applications for DNA for their own sake – makes it a threat both to public safety as well as to corporate profits.

For the moment, we hasten to add, the threat seems more imagined than real. As Wohlsen puts it, “…bad guys with a semester of community college biology under their belts can get far more destruction for their dollar by whipping up a vat of botulism-causing bacteria in their basements than by trying to splice genes.”

Government meddling may be enough to slow the pace of biohacking. But I hope that does not stop it. Just as free-flowing, non-establishment creativity has helped give us Linux, SETI and the remarkable political power of Twitter and other social networks, DNA hacking may turn out to be a potent source for good.

This will only happen before a couple of those obstacles are overcome. First off, cost is a much bigger factor in home-brewed biology than it has ever been in computing. Second, assuming that some of DIY DNA’s discoveries would ultimately have to be converted into mundane intellectual property (IP) even to be applied effectively in the developing world, let alone in developed countries, the “quick-and-dirty” approach might well be too “dirty” to ever become the underlying IP for a biotech or diagnostics startup. It is no coincidence that a typical biotech company raises $100 million or more before it achieves a viable proof of concept for a new therapeutic. And that is just the beginning.

The best science writing reads like science fiction, introducing people, techniques and discoveries right now that make us feel like the future has arrived and it’s even shinier and newer than we thought it would be. Although neither a critical discovery nor a galvanizing leader has emerged from this potent stew, Biopunk succeeds in thought-provokingly preparing us for the new world that will greet us when they do.

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*Steampunk photo courtesy Curious Expeditions under Creative Commons license

Further reading: DIY DNA in art and science fiction

(Special thanks to EW for these recommendations)

Margaret Atwood’s provocative novels Oryx and Crake and The Year of the Flood explore some possible and frightening futures.

Strange Culture is an indie film chronicling the strange story, also retold in Biopunk, of artist and professor Steve Kurtz who, according to the Netflix plot summary “on the eve of his new exhibit, was shocked by the news that his wife had died of heart failure. The medics on the scene became suspicious of Kurtz’s artistic media, which includes genetically modified foods, and the FBI accused him of bioterrorism.”

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