Aspirin is the gold standard for antiplatelet therapy and a daily low-dose aspirin is widely prescribed for the prevention of cardiovascular disease.
Now, a new study suggests that common genetic variation in the gene for catechol-O-methyltransferase (COMT) may modify the cardiovascular benefit of aspirin and, in some people, may confer slight harm. The findings, from Harvard Medical School investigators at Beth Israel Deaconess Medical Center and Brigham and Women’s Hospital, are published in the American Heart Association journal Arteriosclerosis, Thrombosis, and Vascular Biology.
“This is one of the few cases where you can identify a single genetic polymorphism which has a significant interaction with aspirin such that it affects whether or not it protects against cardiovascular disease,” said first author Kathryn Hall, an HMS research fellow and investigator in the Division of General Medicine and Primary Care at Beth Israel Deaconess.
COMT is a key enzyme in the metabolism of catecholamines, a group of hormones that include epinephrine, norepinephrine and dopamine. These hormones are implicated in a broad spectrum of disorders, including hypertension.
“We were initially interested in finding out if the COMT gene affected people’s susceptibility to cardiovascular disease, such as myocardial infarction or ischemic stroke,” Hall said.
Knowing that aspirin is commonly prescribed for the prevention of cardiovascular disease, the investigators also wanted to learn if genetic variation in COMT would influence aspirin’s potential benefit.
To answer these questions, the researchers used data from the Women’s Genome Health Study, a cohort of more than 23,000 women who were followed for 10 years in a randomized double-blind, placebo-controlled trial of low-dose aspirin or vitamin E for the primary prevention of cardiovascular disease. Their analysis focused on val158met, a common variant in the COMT gene. Individuals who have two copies of the gene for the enzyme’s high-activity valine form, the “val/vals,” have been shown to have lower levels of catecholamines compared to individuals who have two copies of the gene for the enzyme’s low-activity methionine form, the “met/mets.” The val/met people are in between.
“When we examined women in the placebo arm of the trial, we found that the 23 percent of the women who were ‘val/vals’ were naturally protected against cardiovascular disease,” said senior author Daniel Chasman, HMS associate professor of medicine at Brigham and Women’s. He is also a genetic epidemiologist in the Division of Preventive Medicine at the hospital. “This finding, which was replicated in two other population-based studies, was in itself of significant interest.”
The investigation further revealed the surprising discovery that when the women with the val/val polymorphism were allocated to aspirin, this natural protection was eliminated.
“As we continued to look at the effects of drug allocation, we found that val/val women who were randomly assigned to aspirin had more cardiovascular events than the val/vals who were assigned to placebo,” says Chasman. Among the 28 percent of women who were met/met, the opposite was true, and these women had fewer cardiovascular events when assigned to aspirin compared to placebo. The benefit of aspirin compared to placebo allocation for met/mets amounted to reduction of one case of incident cardiovascular disease for 91 treated women over 10 years of study follow-up. By contrast, the harm of aspirin compared to placebo allocation for the val/val women was an increase of one case per 91 treated.
The researchers further found that rates of cardiovascular disease were also reduced in met/met women assigned to vitamin E compared to those assigned to placebo.
The authors stressed that the findings will require further research and replication to understand their potential for clinical impact. Nonetheless, they note that because aspirin is preventively prescribed to millions of individuals and the COMT genetic variant is extremely common, this study underscores the potential importance of individualizing therapies based on genetic profiles.
“What this study suggests is that we can be smarter about the groups of patients that would most likely benefit from aspirin,” said study coauthor Joseph Loscalzo, chairman of the Department of Medicine and physician-in-chief at Brigham and Women’s. He is also the Hersey Professor of the Theory and Practice of Physic at HMS. “Rather than give aspirin to all patients with risk factors for heart disease, we need to use modern genomics and genetics to identify those individuals for whom aspirin has the greatest benefit and the lowest risk of adverse effects.”
One possible reason for the val/val protection could lie in COMT’s role in the breakdown of epinephrine, the “fight or flight” hormone, which is tightly linked to regulation of the cardiovascular system.
“When epinephrine levels rise in response to stress, blood pressure goes up and high blood pressure is a precursor to heart disease,” said Hall. “One possibility is that val/val individuals have less epinephrine than met/met individuals because their COMT is more efficient at breaking it down. This might help to naturally protect them against cardiovascular disease—that’s our working hypothesis. It’s harder to explain why the effect is modified by aspirin and that’s what we’re in the lab aggressively trying to figure out.”
The Women’s Genome Health Study is supported by HL043851 and HL080467 from the National Heart, Lung, and Blood Institute and CA 047988 from the National Cancer Institute. This study was also supported by NIH grants T32A5000051; R01AT004662; K24AT004095; R21AT002860; 3R01AT004662-02S1from the National Center for Complementary and Alternative Medicine.
Adapted from a Beth Israel Deaconess news release.
A next-generation genome-editing system developed by Harvard Medical School investigators at Massachusetts General Hospital substantially decreases the risk of producing unwanted, off-target gene mutations. In a paper published in Nature Biotechnology, the researchers report a new CRISPR-based RNA-guided nuclease technology that uses two guide RNAs, significantly reducing the chance of cutting through DNA strands at mismatched sites.
“This system combines the ease of use of the widely adopted CRISPR/Cas system with a dimerization-dependent nuclease activity that confers higher specificity of action,” said J. Keith Joung, HMS associate professor of pathology at Mass General and senior author of the report. “Higher specificity will be essential for any future clinical use of these nucleases, and the new class of proteins we describe could provide an important option for therapeutic genome editing.”
Engineered CRISPR-Cas nucleases are genome-editing tools that combine a short RNA segment matching its DNA target with a DNA-cutting enzyme called Cas9. The tools have been the subject of much investigation since their initial development in 2012. Easier to use than the earlier zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) systems, they have successfully induced genomic changes in several animal model systems and in human cells. But in a previous Nature Biotechnology paper published in June 2013, Joung’s team reported that CRISPR-Cas nucleases could produce additional mutations in human cells, even at sites that differed from the DNA target by as much as five nucleotides (the subunits of DNA).
To address this situation, the investigators developed a new platform in which the targeting function of Cas9 was fused to a nuclease derived from a well-characterized enzyme called Fokl, which functions only when two copies of the molecule are paired, in a relationship called dimerization. This change essentially doubled the length of DNA that must be recognized for cleavage by these new CRISPR RNA-guided Fokl nucleases, significantly increasing the precision of genome editing in human cells. Joung and his colleagues also demonstrated that these new RNA-guided Fokl nucleases are as effective at on-target modification as existing Cas9 nucleases that target a shorter DNA sequence.
“By doubling the length of the recognized DNA sequence, we have developed a new class of genome-editing tools with substantially improved fidelity compared with existing wild-type Cas9 nucleases and nickases,” the enzymes that cleave a single DNA strand, said Joung, who is associate chief for research in the Mass General Department of Pathology.
The research team also has developed software that enables users to identify potential target sites for these new RNA-guided Fokl nucleases. The freely available software package is ZiFiT Targeter.
The study was supported by National Institutes of Health Director’s Pioneer Award DP1 GM105378; NIH grants R01 GM088040, P50 HG005550 and R01 AR063070; and the Jim and Ann Orr Massachusetts General Hospital Research Scholar Award. Joung is a co-founder of Editas Medicine Inc., which has an exclusive option to license the new CRISPR RNA-guided Fokl nuclease technology for therapeutic applications.
Adapted from a Mass General news release.
Inherited mutations in the BRCA1 or BRCA2 tumor suppressor genes are by far the most frequent contributors to hereditary cancer risk in the human population, often causing breast or ovarian cancer in young women of child-bearing age. Attempts to test the role that the BRCA genes play in regulating a repair process associated with genome duplication have proven frustratingly difficult in living mammalian cells.
Now Harvard Medical School investigators at Beth Israel Deaconess Medical Center report a new mechanism by which BRCA gene loss may accelerate cancer-promoting chromosome rearrangements. The new findings explain how the loss of BRCA1 or BRCA2 function impairs homologous recombination, a normally accurate repair process used to fix DNA breaks, and actually stimulates faulty, error-prone homologous recombination repair.
Described online in the April 28 issue of Nature, the discovery could ultimately provide clinicians with valuable new information to help them ascertain risk and guide patient treatment when faced with BRCA mutations of uncertain significance. The finding also offers a potentially valuable new tool for the development of cancer therapeutics.
“Mutations in the BRCA genes cause breast and ovarian cancers that affect thousands of women throughout the U.S. and around the world, often striking them in the prime of life,” said senior author Ralph Scully, HMS associate professor of Medicine at Beth Israel Deaconess in the hospital’s Breast Cancer Oncology program. “For almost two decades, scientists have been striving to better understand the tumor suppressor functions of BRCA1 and BRCA2.”
Potentially harmful breaks in DNA strands commonly occur during DNA replication, a prerequisite for cell division. These breaks occur when the replication fork that duplicates the genome stalls at sites of DNA damage. If not properly repaired, the breaks can promote genomic instability, leading to cancer and other diseases.
“Some years ago, we and others suggested that BRCA1 and BRCA2 regulate homologous recombination at sites of stalled replication,” explains Scully. “We believe that this function is critical to how these genes suppress breast and ovarian cancer. Until now, we haven’t had the tools necessary to study in molecular detail the homologous recombination processes at sites of replication fork stalling in the chromosomes of a living mammalian cell.”
To solve this problem, first author Nicholas Willis, HMS research fellow in medicine in the Scully laboratory, created a new tool by harnessing a protein-DNA complex that evolved in bacteria.
“We found that the Escherichia coli Tus/Ter complex can be engineered to induce site-specific replication fork stalling and chromosomal homologous recombination in mouse cells,” explained Willis. “In its essence, E. coli bacteria—a standard model organism in science—has evolved a very simple system to arrest replication forks in a site-specific manner.”
This system is composed of short DNA elements called Ter sites, which are 21 to 23 base pairs long and tightly bound by the protein Tus. “Tus binds these Ter elements with extremely high affinity and, upon replication fork approach, acts as a barrier to fork progression along the DNA. Tus/Ter effectively sets up a ‘roadblock’ and stalls the replication fork.”
The acid test for the new tool, said the authors, came when this same short Ter sequence was placed into a reporter, a slightly larger DNA sequence that can undergo certain rearrangements within a chromosome when triggered to do so. “When it engaged in homologous recombination, a change in the sequence caused the cells to express green fluorescent protein,” said Willis. “When the cells glowed green, we knew we had a positive event.”
The team adapted the reporter sequence to distinguish between error-free/high-fidelity homologous recombination and an error-prone/aberrant form of homologous recombination. “Remarkably, when we studied cells lacking BRCA1 or BRCA2, we found that the frequency of aberrant homologous recombination events triggered at Tus/Ter-stalled replication forks had actually increased compared to normal cells. We knew at this point that we had discovered a new and important process by which BRCA gene loss promotes cancer.”
The discovery provides a promising bridge between basic science and the clinic, said Scully. “Sometimes a genetic sequencing test reveals a mutation in BRCA1 or BRCA2 that has not been definitively associated with cancer,” he said. Often described as “variants of uncertain significance,” these mutations are not found in high enough frequency in healthy women or in women with breast or ovarian cancers to allow the specific BRCA1 or BRCA2 mutations to be reliably classified as high risk or low risk. This is an important issue because a woman with a known high-risk BRCA gene mutation may elect to undergo potentially lifesaving prophylactic mastectomy or oophorectomy, Scully said.
“There is a growing appreciation that careful measurement of the homologous recombination functions of BRCA1 and BRCA2 variants-of-uncertain-significance mutants might help to classify them into high-risk or low-risk groups,” he said. “It would be gratifying if our system could contribute new information to help ongoing efforts to classify these mutants.”
Furthermore, understanding the mechanisms that regulate homologous recombination at stalled replication forks might hold additional promise for the development of novel cancer therapeutics. “If we could use this tool to help develop new cancer therapies, it would be a grand slam,” said Scully. “This new system might also be useful in genome editing, which is considered a groundbreaking technology used for the development of new gene therapies.”
This study was funded, in part, by National Institutes of Health grants R01CA095175, R01GM073894, R21CA144017, and R37GM26938; by National Institutes of Health /National Cancer Institute postdoctoral fellowship 5T32CA081156; and by ACS postdoctoral fellowship PF-12-248-01-DMC.
Adapted from a Beth Israel Deaconess news release.
The humble aspirin may have another beneficial effect beyond easing the pain of headache and reducing the risk of heart attack: lowering colon cancer risk among people with high levels of a specific enzyme.
The finding comes from a multi-institutional team that analyzed data and samples from two long-term studies involving nearly 128,000 participants. The researchers discovered that people who took aspirin and had high levels of a specific enzyme in their colons had half the risk of developing colorectal cancer compared with people who also took aspirin but whose colons showed low levels of the enzyme, called 15-hydroxyprostaglandin dehydrogenase, or 15-PGDH for short. About half of the population possesses high levels of 15-PGDH.
The results appear in the April 23 edition of Science Translational Medicine. While previous trials and prospective studies have shown that aspirin use reduces colorectal cancer risk, this retrospective study may explain why aspirin benefits some people, but not others.
The research team included scientists from Brigham and Women’s Hospital, Case Western Reserve School of Medicine, Dana-Farber Cancer Institute, Harvard Medical School, Massachusetts General Hospital and University Hospitals Case Medical Center.
“If you looked at the folks from the study who had high 15-PGDH levels and took aspirin, they cut their risk of colon cancer by half,” said senior author Sanford Markowitz, Ingalls Professor of Cancer Genetics at Case Western Reserve School of Medicine. “If you looked at the folks from the study who were low for 15-PGDH, they did not benefit at all from taking aspirin. These findings represent a clean Yes-No about who would benefit from aspirin.”
According to the American Cancer Society, colorectal cancer is the second leading cause of cancer-related deaths in the United States, with predictions that 137,000 Americans will develop the disease and 50,000 will die from it in 2014.
In the current study, the scientists built on earlier research indicating that regular use of non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, reduces the risk of developing colon cancer for some but not all individuals. Markowitz, also a medical oncologist at University Hospitals Case Medical Center, joined co-senior author Andrew Chan, HMS associate professor of medicine and a gastroenterologist at Mass General, to explore whether the presence of 15-PGDH led to different outcomes in developing colon cancer.
They hope to develop a test to guide physicians and patients in determining whether aspirin would help them.
The team examined tissues of 270 colon cancer patients who were among 127,865 participants followed for more than 30 years in the Harvard-based Nurses’ Health Study and Health Professionals Follow-up Study. Previous reports from the Mass General and Dana-Farber team showed that participants in these studies who regularly took aspirin had a lower risk of colorectal cancer. In earlier research, Case Western Reserve investigators and Monica Bertagnolli, HMS professor of surgery at Brigham and Women’s, had found that 15-PGDH appeared to enhance the ability of celecoxib, an anti-inflammatory medication commonly known as Celebrex, to prevent colon tumors in mice and in 16 humans tested. But when 15-PGDH was low or absent, celecoxib did not prevent colon tumors in mice or humans.
In the latest study, the investigators combined forces in a larger study to examine whether 15-PGDH levels might also be associated with the colon cancer-preventing benefits of aspirin, which lacks the adverse cardiovascular side effects of celecoxib.
The Mass General and Dana-Farber team dissected normal colon tissue from the pathology specimens of participants who developed colon cancer over the studies’ follow-up periods. The team at Case Western Reserve then analyzed these colon tissues to identify which among them had high or low levels of colon 15-PGDH. The investigators at Mass General and Dana-Farber examined how participants’ aspirin use and levels of 15-PGDH might be related to the risk of colorectal cancer.
The study’s results could lead to a test that would allow more personalized decisions about treatment to prevent colorectal cancer. People whose 15-PGDH levels indicate aspirin would have little benefit might choose to avoid the potential gastrointestinal side effects—such as stomach ulcers—that can accompany aspirin use. The researchers plan to develop a cost-effective and accessible test for measuring 15-PGDH in the colon. Chan and Markowitz believe such a test could become part of current medical practice.
“During a colonoscopy, a gastroenterologist could easily and safely take an additional biopsy from the colon in individuals for whom preventive aspirin treatment might be appropriate,” Chan said.
“There would be no reason why a good hospital pathology laboratory could not establish the test for 15-PGDH,” Markowitz said.
To confirm their findings, the study authors hope to launch a randomized, prospective clinical trial in which high-risk patients would be identified, treated with aspirin or a placebo, and monitored for development of colorectal tumors.
The mechanisms of action in 15-PGDH and in aspirin make them key players in colon cancer, the scientists said. Prostaglandins promote development of colon cancer. Aspirin helps prevent colon cancer development by blocking prostaglandins from being generated, while 15-PGDH helps prevent colon cancer development by catalyzing a reaction that “chews up” prostaglandins. Markowitz refers to the gene that produces 15-PGDH as the body’s genetic form of aspirin. The study suggests that both aspirin and 15-PGDH must work together to effectively prevent colon cancer, bringing the most benefit to individuals who have high levels of 15-PGDH.
“This study highlights the benefits of the relatively new practice of molecular pathological epidemiology, ” said co-senior author Shuji Ogino, HMS associate professor of pathology at Brigham and Women’s and Dana-Farber. “The molecular pathology part relates to analysis of 15-PGDH gene expression level in the normal colon to classify cancer based on molecular pathogenesis, while the epidemiology part relates to collection and analysis of aspirin use data in a population. This is an integration of these analyses."
Other researchers involved in the study include first author Stephen Fink, an instructor at Case Western Reserve; co-authors Mai Yamauchi and Reiko Nishihara, HMS research fellows at Dana-Farber; and senior author Charles S. Fuchs, HMS professor of medicine at Dana-Farber.
The study was supported by the Entertainment Industry Foundation’s National Colorectal Cancer Research Alliance; the National Cancer Institute’s GI-SPORE program (Specialized Programs of Research Excellence in Gastrointestinal Cancers) and Early Detection Research Network; National Institutes of Health grants P01 CA87969, P01 CA55075, 1UM1 CA167552, R01 CA136950, P50 CA127003, R01 CA151993, P50 CA150964, U01 CA152756, R01 CA137178 and K24 DK 098311; the Damon Runyon Cancer Research Foundation; and gifts from the Cleveland-based Marguerite Wilson Foundation, the Leonard and Joan Horvitz Foundation, the Richard Horvitz and Erica Hartman-Horvitz Foundation, and the Boston-based Bennett Family Fund for Targeted Therapies Research.
Adapted from a joint Case Western Reserve and Mass General news release.
If you haven’t thought about reworking the human genome so people can colonize other planets, don’t worry. Plenty of people are on it.
Scientists of many stripes have been figuring out what barriers would keep us from calling distant, inhospitable galactic real estate “home” if—or when, depending on your point of view— we damage the Earth enough to face extinction. And then there’s the whole question of whether we should try to win a stay of execution for our species. After all, what makes us so special?
Those questions were just the beginning of a free-form symposium hosted March 13 by the HMS Department of Genetics on “Genetics, Biomedicine, and the Human Experience in Space,” the standing-room-only crowd in attendance fueled by pizza and unbridled curiosity.
Speakers quickly made clear why space travel and exploration over vast, uncharted distances depends on numerous, unknown factors hidden in our genes. Living with microgravity while being bombarded with cosmic rays can affect different people different ways. Scientists want to know why—and which genes might make it better or worse.
Also, space is just cool.
The Role of Genetics
The session unleashed uninhibited discussion, with a fairly even split between prepared presentations and informed thinking-out-loud improv from the audience.
“We are a medical school. Whether or not you agree with sending people into space, we are responsible for their health on and off the planet,” said Wu.
Well-known muscle and skeletal weakness and sleep disruption are not the only problems humans encounter in space. Physical concerns ride along with behavioral and neuropsychiatric issues aboard current spacecraft, not to mention whatever vehicles might ferry people farther away. It’s lonely out there.
Thinking about travel to Mars, one of our nearest neighbors, is daunting for robots, much less people. Just ask symposium guest Adam Steltzner, mechanical systems lead at the Jet Propulsion Lab, about the prodigious work that brought back what we know about the planet. Or Dorit Donoviel, deputy chief scientist and industry forum lead of the National Space Biomedical Research Institute, and assistant professor in the Department of Pharmacology and Center for Space Medicine at Baylor College of Medicine, who studies astronauts and the challenges they face, including problems with vision and headaches.
Focusing on Space
HMS geneticists spoke about the intersections between their scientific focus and space. Susan Dymecki said she began thinking about why cosmonauts in the former Soviet Union’s space program were forbidden from playing chess on board space flights. The answer involves aggression and impulsivity.
For HMS professor of genetics David Sinclair, this intersection involves the potential advantages of extant human variation and rallying our genetics to counter aging during long-distance travel spanning hundreds of thousands of years.
Bruce Yankner, HMS professor of genetics, talked about protecting the brain and memory in space. Wu presented her vision for using ultraconserved elements, which some consider to be among the most mysterious sequences of the human genome, to orchestrate chromosome behavior
to and thus protect genomes against cosmic radiation in space.
Mary Bouxsein, a biomechanical engineer and HMS assistant professor of orthopedic surgery at Beth Israel Deaconess Medical Center, a last minute addition to the program, showed the devastating effects of space flight on bone, and how that might be prevented in space—and on Earth -- with a newly developed therapy.
Genetics professor Gary Ruvkun, whose talk was entitled “What’s true for E. coli is true for the elephant” and our speculative kin on Gliese 667 Cf, (a potential Class M planet in the Gliese 667 star system), peppered the meeting with a positive view of the extinction of the human species and then proposed that, rather than travel to another planet, we “print” ourselves there. Conversely, we could print extraterrestial life on Earth.
George Church, the Robert Winthrop Professor of Genetics, suggested using genomics to identify and engage human protective variants, speculating that, by ridding ourselves of our microbiome and taking advantage of variants that suppress pain, we might create a habitat in which surgeries can occur without anesthesia or need for sterilization.
Space is vast, cold and hard for us humans, and outside of Earth, its planets, and moons—too hot, too cold, too toxic for life that evolved here— are not much more welcoming. Should we want to go there, and decide who is best suited to do so, a great deal more work needs to be done not only in jet propulsion but in human genetics.
In biology, as in real estate, location matters. Working copies of active genes -- called messenger RNAs or mRNAs -- are positioned strategically throughout living tissues, and their location often helps regulate how cells and tissues grow and develop. But to analyze many mRNAs simultaneously, scientists have had to grind cells to a pulp, which left them no good way to pinpoint where those mRNAs sat within the cell.
Now a team at the Wyss Institute of Biologically Inspired Engineering at Harvard University and Harvard Medical School, in collaboration with the Allen Institute for Brain Science, has developed a new method that allows scientists to pinpoint thousands of mRNAs and other types of RNAs at once in intact cells -- all while determining the sequence of letters, or bases, that identify them and reveal what they do.
The method, called fluorescent in situ RNA sequencing (FISSEQ), could lead to earlier cancer diagnosis by revealing molecular changes that drive cancer in seemingly healthy tissue. It could track cancer mutations and how they respond to modern targeted therapies, and uncover targets for safer and more effective ones.
The method could also help biologists understand how tissues change subtly during embryonic development -- and even help map the maze of neurons that wire the human brain. The researchers reported the method in today’s online edition of Science.
“By looking comprehensively at gene expression within cells, we can now spot numerous important differences in complex tissues like the brain that are invisible today,” said George Church, a professor of genetics at Harvard Medical School and a core faculty member at the Wyss Institute. “This will help us understand like never before how tissues develop and function in health and disease.”
Locking RNAs in Place
Healthy human cells typically turn on nearly half of their 20,000 genes at any given time, and they choose those genes carefully to produce the desired cellular responses. Moreover, cells can dial gene expression up or down, adjusting to produce anywhere from a few working copies of a gene to several thousand.
But simultaneously pinpointing the cellular location of all those mRNAs is a tall order.
Church and Je Hyuk Lee, a research fellow at Harvard Medical School and the Wyss Institute, were up for the challenge. Moreover, they wanted to simultaneously determine the sequence of those RNAs, which identifies them and often reveals their function.
Lee and his colleagues first treated the tissue chemically to fix the cell’s thousands of RNAs in place. Then they used enzymes to copy those RNAs into DNA replicas, and copy those replicas many times to create a tiny ball of replica DNA fixed to the same spot.
They managed to fix and replicate thousands of the cell’s RNAs at once -- but then became a victim of their own success. The RNAs were so tightly packed inside the cell that even a tricked-out microscope and camera could not distinguish the flashing lights of one individual ball of replica DNA from those of its neighbors.
A new way to image RNA
To solve that problem, the researchers pioneered an unconventional method to visualize tiny objects inside cells. It works like an urban postal system. If a postmaster tried to identify each home in her city by color, she would quickly run out of colors as new homes were built, resulting in undelivered mail. Instead, postmasters keep track of each home by assigning it a unique address.
The researchers realized they could assign each RNA in the cell a unique address: the sequence of “letters,” or bases, in the RNA molecule itself. They figured they could read the address using methods akin to next-generation DNA sequencing, a set of high-speed genome sequencing methods Church helped develop in the early 2000s.
In next-gen sequencing, scientists grind up tissue, extract its DNA, break the DNA into pieces, then dilute those pieces enough so that each piece of DNA sticks to a separate spot on a glass slide. They use enzymes and four different fluorescent dyes -- one each for each of the four “letters,” or bases -- to make the DNA flash a sequence of colors that reveals its sequence.
By analogy, the scientists sought to fix RNA in place in the cell, make a tiny ball with many matching DNA replicas of each RNA, then adapt next-gen DNA sequencing so it worked in fixed cells. The four flashing colors would reveal the base sequence of each replica DNA, which would tell them the base sequence of the matching RNA from which it was derived. And those sequences would in theory provide an unlimited number of unique addresses – one for each of the original RNAs.
The scientists struggled at first to visualize the flashing lights of individual balls of replica DNA from a distance where the whole landscape of the tissue remained in view.
They succeeded by selectively turning on just a fraction of those flashing dots at any given time, so they could distinguish single balls of replica DNA flashing across the cellular landscape.
The strategy would only work, however, if they could actually read enough of the base sequence to provide a unique address. At first they could not determine more than six bases in the replica DNA, which did not provide enough unique addresses to identify individual genes in the human genome. That’s when Evan Daugharthy, a graduate student at Harvard Medical School, stepped in.
FISSEQ at Work
Daugharthy first devised an algorithm to locate the sequence of the replica DNA with the known sequence of genes in the human genome. Flashing lights that did not correspond to a real gene were erased from the image.
Then Daugharthy hacked a commercial DNA sequencing kit, which enabled the team to sequence 30 bases, more than enough to provide each replica DNA with a unique address. In this way the team could create a composite image representing the sequence, and location, of RNA corresponding to every gene in the human genome.
Lee, Daugharthy and their colleagues then tested the method to detect the genes skin cells turn on as they multiply and migrate to heal a simulated wound in a petri dish. Cells growing into the wound had 12 genes that were activated much more or much less than nearby cells sitting idly on the sidelines. Similar experiments could identify new markers of diseased tissue or new targets for targeted molecular therapies.
“What George’s team has accomplished is a technological tour de force,” said Wyss Institute founding director Don Ingber. “By spotting incredibly subtle but incredibly important changes in gene expression and precisely defining their position inside the cell, they have helped open the door to a new age of cellular diagnostics.”
The work was funded by the National Institutes of Health, the Allen Institute for Brain Science and the Wyss Institute.
A new microscopy method could enable scientists to generate snapshots of dozens of different biomolecules at once in a single human cell, a team from the Wyss Institute of Biologically Inspired Engineering at Harvard University reported Sunday in Nature Methods.
Such images could shed light on complex cellular pathways and potentially lead to new ways to diagnose disease, track its prognosis, or monitor the effectiveness of therapies at a cellular level.
Cells often employ dozens or even hundreds of different proteins and RNA molecules to get a complex job done. As a result, cellular job sites can resemble a busy construction site, with many different types of these tiny cellular workers coming and going. Today's methods typically only spot at most three or four types of these tiny workers simultaneously. But to truly understand complex cellular functions, it's important to be able to visualize most or all of those workers at once, said Peng Yin, assistant professor of systems biology at Harvard Medical School and a core faculty member at the Wyss Institute.
"If you can see only a few things at a time, you are missing the big picture," Yin said.
Yin's team sought a way to take aerial views of job sites that could spot up to dozens of types of biomolecules that make up large cellular work crews.
To capture ultrasharp images of biomolecules, they had to overcome laws of physics that stymied microscopists for most of the last century. When two objects are closer than about 200 nanometers apart — about one five-hundredth the width of a human hair — they cannot be distinguished using a traditional light microscope: the viewer sees one blurry blob where in reality there are two objects.
Since the mid-1990s, scientists have developed several ways to overcome this problem using combinations of specialized optics, special fluorescent proteins or dyes that tag cellular components.
Ralf Jungmann, now a postdoctoral Fellow working with Yin at the Wyss Institute and Harvard Medical School, helped develop one of those super-resolution methods, called DNA-PAINT, as a graduate student. DNA-PAINT can create ultrasharp snapshots of up to three cellular workers at once by labeling them with different colored dyes.
To visualize cellular job sites with crews of dozens of cellular workers, Yin's team, including Jungmann, Maier Avendano, M.S., a graduate student at Harvard Medical School, and Johannes Woehrstein, a postgraduate research fellow at the Wyss Institute, modified DNA-PAINT to create a new method called Exchange-PAINT.
Exchange-PAINT relies on the fact that DNA strands with the correct sequence of letters, or nucleotides, bind specifically to partner strands with complementary sequences. The researchers label a biomolecule they want to visualize with a short DNA tag, then add to the solution a partner strand carrying a fluorescent dye that lights up only when the two strands pair up. When that partner strand binds the tagged biomolecule, it lights up, then lets go, causing the biomolecule to "blink" at a precise rate the researchers can control. The researchers use this blinking to obtain ultrasharp images.
They then repeat the process to visualize a second target, a third, and so on. Then they overlay the resulting images to create a composite image in which each biomolecule — each cellular worker — is assigned a different color. This allows them to create false-color images that simultaneously show many types of biomolecules — far more than they could simultaneously visualize by labeling them with different colored dyes. And these false-color images allow them to spot enough cellular workers at once to capture the entire scene.
To test Exchange-PAINT, the researchers created 10 unique pieces of folded DNA, or DNA origami, that resembled the numerals 0 through 9. These numerals could be resolved with less than 10 nanometers resolution, or one-twentieth of the diffraction limit.
The team was able to use Exchange-PAINT to capture clear images of the 10 different types of miniscule DNA origami structures in one image. They also used the method to capture detailed, ultrasharp images of fixed human cells, with each color tagging an important cellular component — microtubules, mitochondria, Golgi apparatus, or peroxisomes.
Yin expects the method, with further development, to be able to visualize dozens of cellular components at once.
"Peng's exciting new imaging work gives biologists an important new tool to understand how multiple cellular components work together in complex pathways," said Wyss Institute Founding Director Don Ingber. "I expect insights from those experiments to lead to new ways to diagnose and monitor disease." Ingber is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children's Hospital and Professor of Bioengineering at Harvard School of Engineering & Applied Sciences.
In addition to Yin, Jungmann, Avendano, and Woehrstein, the team included Mingjie Dai, a graduate student in biophysics at Harvard University and William Shih, a Wyss Institute core faculty member who is also associate professor of biological chemistry and molecular pharmacology at Harvard Medical School and associate professor of cancer biology at the Dana-Farber Cancer Institute. The work was funded by the National Institutes of Health, the Office of Naval Research, the National Science Foundation, the Humboldt Foundation, and the Wyss Institute.
Adapted from a Wyss Institute news release.
A simple adjustment to a powerful gene-editing tool improves its precision, Harvard Medical School researchers at Massachusetts General Hospital report.
In a paper published in Nature Biotechnology, the scientists have shown how adjusting the length of guide RNAs in synthetic enzymes called CRISPR-Cas RNA-guided nucleases can substantially reduce off-target DNA mutations, a limitation the team revealed just last year.
“Simply by shortening the length of the guide RNA targeting region, we saw reductions in the frequencies of unwanted mutations at all of the previously known off-target sites we examined,” said J. Keith Joung, HMS associate professor of pathology at Mass General and senior author of the paper. “Some sites showed decreases in mutation frequency of 5,000-fold or more, compared with full-length guide RNAs. Importantly, these truncated guide RNAs—which we call tru-gRNAs—are just as efficient as full-length gRNAs at reaching their intended target DNA segments.”
Last year two groups reported their success in using a tool borrowed from a bacterial immune system called Cas, short for CRISPR-associated systems, which in turn stands for Clustered Regularly Interspaced Short Palindromic Repeats. In bacteria the Cas9 enzyme system uses short stretches of RNA to target and then cut invading viral DNA. Scientists have customized this system to work in human cells, creating an RNA-guided editing tool that allows them to integrate DNA changes into the genomes of living cells.
Later last year Joung’s team found that in human cells, CRISPR-Cas RNA-guided nucleases could also cause mutations in DNA sequences with differences of up to five nucleotides from the target, which could seriously limit the proteins’ clinical usefulness. The team followed up with a hypothesis that could seem counterintuitive: Shortening the gRNA segment might reduce off-target mutations.
“Some of our experiments from last year suggested that one could mismatch a few nucleotides at one end of the gRNA complementarity region without affecting the targeting activity,” Joung explained. “That led us to wonder whether removing these nucleotides could make the system more sensitive to mismatches in the remaining sequence.”
The CRISPR-Cas RNA-guided nucleases most widely used by researchers include a 20-nucleotide targeting region within the gRNA. To test their theory, the team constructed RNA-guided nucleases with progressively shorter gRNAs. They found that while gRNAs with targeting segments of 17 or 18 nucleotides were at least as efficient as full-length gRNAs in reaching their targets, those with 15- or 16-nucleotide targeting segments had reduced or no targeting activity. Subsequent experiments found that 17-nucleotide truncated RNA-guided nucleases efficiently induced the desired mutations in human cells, with greatly reduced or undetectable off-target effects, even at sites with only one or two mismatches.
“While we don’t fully understand the mechanism by which tru-gRNAs reduce off-target effects, our hypothesis is that the original system might have more energy than it needs, enabling it to cleave even imperfectly matched sites,” Joung said. “By shortening the gRNA, we may reduce the energy to a level just sufficient for on-target activity, making the nuclease less able to cleave off-target sites. But more work is needed to define exactly why tru-gRNAs have reduced off-target effects.”
Joung’s team has incorporated this capability for finding tru-gRNA target sites into ZiFiT Targeter, a freely available software package designed to identify potential target sites for several DNA-editing technologies.
The study was supported by National Institutes of Health Director’s Pioneer Award DP1 GM105378, NIH grants R01 GM088040 and P50 HG005550, and the Jim and Ann Orr MGH Research Scholar Award.
Adapted from a Mass General news release.
A new technology developed by Harvard Medical School researchers at the Massachusetts General Hospital Center for Systems Biology allows the simultaneous analysis of hundreds of cancer-related protein markers from minuscule patient samples gathered through minimally invasive methods. This powerful and sensitive technology uses antibodies linked to unique DNA “barcodes” to detect a wide range of target proteins.
It could serve as a tool to help clinicians gain insights into the biology of cancer progression as well as determine why certain cancer therapies stop working or are ineffective to begin with. Development of the technology is reported in Science Translational Medicine.
Minimally invasive techniques—such as fine-needle aspiration or circulating tumor cell analysis—are increasingly employed to track treatment response over time in clinical trials, as the tests can be simple and cheap to perform. Fine-needle aspirates are also much less invasive than core biopsies or surgical biopsies, since very small needles are used. The challenge has been to comprehensively analyze the very few cells that are obtained via this method.
“What this study sought to achieve was to vastly expand the information that we can obtain from just a few cells,” explained Cesar Castro, HMS instructor in medicine at Mass General and a co-author of the paper. “Instead of trying to procure more tissue to study, we shrank the analysis process so that it could now be performed on a few cells.”
Until now, pathologists have been able to examine only a handful of protein markers at a time for tumor analyses. With this new technology, the researchers have demonstrated the ability to look at hundreds of markers simultaneously, down to the single-cell level.
“We are no longer limited by the scant cell quantities procured through minimally invasive procedures,” Castro said. “Rather, the bottleneck will now be our own understanding of the various pathways involved in disease progression and drug target modulation.”
The new method uses an approach known as DNA-barcoded antibody sensing, in which unique DNA sequences are attached to antibodies against known cancer marker proteins. The DNA “barcodes” are linked to the antibodies with a special type of glue that breaks apart when exposed to light. When mixed with a tumor sample, the antibodies seek out and bind to their targets; then a light pulse releases the unique DNA barcodes of these bound antibodies that are subsequently tagged with fluorescently labeled complementary barcodes. The tagged barcodes can be detected and quantified via imaging, revealing which markers are present in the sample.
After initially demonstrating and validating the technique’s feasibility in cell lines and single cells, the team tested it on samples from patients with lung cancer. The technology was able to reflect the great heterogeneity—differences in features such as cell-surface protein expression—of cells within a single tumor and to reveal significant differences in protein expression between tumors that appeared identical under the microscope. Examination of cells taken at various time points from participants in a clinical trial of a targeted therapy drug revealed patterns that distinguished those who did and did not respond to treatment.
“We showed that this technology works well beyond the highly regulated laboratory environment, extending into early-phase clinical trials,” said Castro, who is also a medical oncologist in the Mass General Cancer Center and director of the Cancer Program within the hospital’s Center for Systems Biology. “In this era of personalized medicine, we could leverage such technology not only to monitor but actually to predict treatment response. By obtaining samples from patients before initiating therapy and then exposing them to different chemotherapeutics or targeted therapies, we could select the most appropriate therapy for individual patients.”
Ralph Weissleder, the HMS Thrall Family Professor of Radiology at Mass General, is corresponding author of the Science Translational Medicine paper. The study was funded in part by National Cancer Institute grant U54 CA15188.
Adapted from a Mass General news release.
A gene mutation associated with several types of cancer may also be responsible for a rare but debilitating brain tumor called papillary craniopharyngioma, according to a team led by HMS investigators from Massachusetts General Hospital, Brigham and Women’s Hospital, Dana-Farber Cancer Institute and the Broad Institute. The discovery, reported in Nature Genetics, could lead to new therapies for this currently hard-to-treat tumor.
“We were delighted to find that the same BRAF mutation previously described in melanomas and other brain tumors appears to be driving the growth of these tumors,” said Priscilla Brastianos, HMS instructor in medicine at Mass General, a research associate at the Broad Institute and co-corresponding author of the paper. “BRAF inhibitors have shown great promise in treating patients with other tumors with this mutation, and we hope to quickly evaluate these drugs in patients with papillary craniopharyngioma in hopes of reducing the serious consequences of this disease.”
Craniopharyngiomas arise at the base of the skull adjacent to the pituitary gland, the hypothalamus and other critical brain structures. Although they are not inherently aggressive tumors, their location means they can significantly compromise vision and other neurologic and endocrine functions. The tumors cling to brain structures, making surgical removal challenging. Radiation therapy can cause vascular abnormalities or other tumors.
There are two subtypes of craniopharyngiomas: adamantinomatous tumors, which are more common in children, and papillary tumors, usually seen in adults. Recent studies have linked mutations in the cancer-causing gene CTNNB1 with adamantinomatous tumors, but before this study, no information was available about the molecular drivers of the papillary subtype.
In their search for possible mutations associated with papillary tumors, the research team first performed whole exome sequencing of 12 adamantinomatous and three papillary craniopharyngiomas. The CTNNB1 mutation was found in 11 of the 12 adamantinomatous samples. For the first time, the well-known tumor-causing BRAF mutation was identified in all three papillary tumors.
The researchers followed that finding with targeted genotyping of tumor samples from an additional 95 patients. Ninety-four percent of the papillary tumors they tested carried the BRAF mutation, while 96 percent of the adamantinomatous tumors had the CTNNB1 mutation. The investigators also confirmed that both types of tumors had very few other mutations. The BRAF or CTNNB1 mutations were present in all tumor cells, suggesting they occurred early in tumor development.
“There are currently no medical therapies available for craniopharyngiomas, but potent compounds that block BRAF signaling are in hand. So we are very hopeful that these targeted therapies can drastically alter the management of these tumors,” said Sandro Santagata, HMS assistant professor of pathology at Brigham and Women’s and a co-corresponding author of the paper. “Inhibitors of the signaling pathway controlled by CTNNB1 that are currently in clinical trials should be investigated for adamantinomatous tumors, and we’re planning to evaluate the BRAF inhibitors that have had promising results against melanoma for treatment of papillary craniopharyngiomas.”
Along with Brastianos, the co-lead authors of the report are Amaro Taylor-Weiner of the Broad Institute and Peter Manley, HMS instructor in pediatrics at Dana-Farber/Boston Children’s Cancer and Blood Disorders Center. Co-senior authors are Mark Kieran, HMS associate professor of pediatrics at Dana-Farber/Boston Children’s, and Gad Getz, HMS associate professor of pathology at Mass General and the Broad Institute. David Louis, the Benjamin Castleman Professor of Pathology at Mass General, also made significant contributions to the study, as did additional collaborators from Mass General, Brigham and Women’s, Dana-Farber, Johns Hopkins University, the University of Pennsylvania and other institutions.
Support for the study includes grants from Pedals for Pediatrics and the Jared Branfman Sunflowers for Life Foundation for Pediatric Brain and Spinal Cord Research.
Adapted from a Mass General news release.