Genetics

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Alpert Prize Recognizes CRISPR Pioneers

Five scientists share award for elucidating bacterial defense system and recognizing its utility for gene editing

The 2016 Warren Alpert Foundation Prize will be awarded to five scientists for their remarkable contributions to the understanding of the CRISPR bacterial defense system and the revolutionary discovery that it can be adapted for genome editing.

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The recipients are:

  • Rodolphe Barrangou, associate professor in the Department of Food, Bioprocessing and Nutrition Sciences and the Todd R. Klaenhammer Distinguished Scholar in Probiotics Research at North Carolina State University
  • Philippe Horvath, senior scientist at DuPont in Dangé-Saint-Romain, France
  • Jennifer Doudna, the Li Ka Shing Chancellor’s Chair in Biomedical and Health Sciences and professor of molecular and cell biology and of chemistry at the University of California, Berkeley
  • Emmanuelle Charpentier, scientific member and director at the Max Planck Institute for Infection Biology in Berlin and professor at Umeå University in Sweden
  • Virginijus Siksnys, professor, chief scientist and department head at the Institute of Biotechnology at Vilnius University in Lithuania

Barrangou and Horvath established that bacteria protect themselves from being killed by pathogens, such as viruses, using a system called CRISPR (short for clustered regularly interspaced short palindromic repeats) to cut up specific segments of the invading viruses’ DNA.

Building on their findings, Doudna, Charpentier and Siksnys realized that the system could be programmed to zero in on any desired genetic sequence in a broader array of organisms, including humans, and that this purposeful cutting could be used to alter or replace the targeted DNA at will.

Together, these discoveries, which were further refined and expanded by the prize recipients and other researchers, have generated a powerful tool for rapidly determining gene function and have democratized the ability to pursue clinical advances such as correcting genetic defects and designing better drugs by making gene editing faster, easier and cheaper than the technologies available previously.

“The game-changing insights achieved by these five scientists led to a technique that has been swiftly embraced across the globe, altering the way we study and understand eukaryotic genetics and offering enormous potential for developing new gene- and cell-based therapies, including treatment strategies for previously intractable genetic diseases,” said Jeffrey S. Flier, dean of the faculty of medicine at Harvard Medical School and chair of the Warren Alpert Foundation Prize Scientific Advisory Committee.

“Determining biochemically and molecularly how the CRISPR system operates and then demonstrating its potential as a gene editing tool is one of the most important technological advances that has been made during my career,” said Cliff Tabin, head of the Department of Genetics at Harvard Medical School and a member of the Alpert Foundation’s scientific advisory prize committee.

“There are a large number of researchers who have contributed to the development of the CRISPR system and in no small measure share credit for it, but in choosing this year’s Alpert Prize recipients, we recognized those who established the understanding of the system and its potential, which others were then able to perfect as a usable tool,” Tabin added.

The Warren Alpert Foundation Prize recognizes scientists whose research has led to the prevention, cure or treatment of human diseases or disorders and constitutes a seminal scientific finding that holds great promise for ultimately changing our understanding of, or ability to treat, disease.

The late Warren Alpert, a philanthropist dedicated to advancing biomedical research, established the prize in 1987. To date, the foundation has awarded more than $3 million to 54 individuals. Eight honorees have also received a Nobel Prize.

This year’s recipients will share an unrestricted award of $500,000 and will be honored at a symposium at Harvard Medical School on Oct. 6.

“These five scientists have made foundational contributions to a technique that has hit the field of genetics like a lightning bolt,” said Bevin Kaplan, director and vice president of the Warren Alpert Foundation and a member of the Harvard Medical School Board of Fellows. “The nature of this work is unprecedented in its far-reaching potential for future therapeutic advances.”

“We are confident that innumerable lives will be positively touched by this work in the years to come,” Kaplan added. “In this way, these exemplary men and women most definitely embody the spirit of the Warren Alpert Foundation Prize. As always, we anticipate a phenomenal symposium and award ceremony in the fall.”

In their own words

Rodolphe Barrangou“I am absolutely delighted and honored to receive the Warren Alpert Foundation Prize. This is a prestigious award, which I am grateful for, and thrilled to share with my colleagues and collaborators, especially Philippe Horvath, with whom I have shared my entire CRISPR journey. It has been enjoyable to observe firsthand the evolution of the CRISPR field in the past decade, and seeing the field evolve from humble beginnings of CRISPR analysis in dairy cultures to driving the genome-editing craze has been fantastic. We could not have dreamed that trying to understand how bacteria resist viruses would eventually lead to the CRISPR revolution we have witnessed. I am thankful to the Warren Alpert Foundation and to the prize selection committee for their consideration, and I look forward to further serving the CRISPR community.” —Rodolphe Barrangou

Philippe Horvath“Our seminal work, published in Science in 2007, had a significant impact on the scientific community. It really set the stage, opened new research avenues and inspired numerous scientists to investigate further in the CRISPR field. Being awarded with the Warren Alpert Foundation Prize is an immense honor and a distinct privilege that I wish to extend to all my collaborators and colleagues at DuPont.” —Philippe Horvath

Jennifer Doudna“​As an alumna of the HMS graduate program, I am particularly honored to receive this award in recognition of research conducted with my collaborator Emmanuelle Charpentier and our outstanding postdoctoral associates and students. We hope that future students are inspired by the value of curiosity-driven research and the passion for fundamental discovery​ that our work represents.” —Jennifer Doudna

Emmanuelle Charpentier“I am honored to receive this award in recognition of our seminal work published in Nature in 2011 and in Science in 2012, including the discovery of tracrRNA and the delineation of its key role in the targeting and editing of DNA by CRISPR-Cas9. This work is a wonderful example of the importance of basic research, demonstrating its relevance for translational science and important medical applications.” —Emmanuelle Charpentier

Virginijus Siksnys“It is a great privilege to be among such outstanding current and former awardees of the Warren Alpert Foundation. This prize makes my work even more enjoyable and challenging. I am really glad that my research, aimed in the beginning on a very basic question of how bacteria protect themselves against phages, paved the way for the development of novel tools for genome-editing applications.” —Virginijus Siksnys

Previous Alpert Prize recipients

The Warren Alpert Foundation Prize has recognized recipients for discoveries that have impacted a wide spectrum of diseases, including asthma, breast cancer, H. pylori, hepatitis B and HIV/AIDS.

Last year’s honorees were Tu Youyou of the China Academy of Chinese Medical Sciences, who went on to receive the 2015 Nobel Prize in Physiology or Medicine, and Ruth and Victor Nussenzweig of NYU Langone Medical Center for their pioneering discoveries in chemistry and parasitology and personal commitments to translate these into effective chemotherapeutic and vaccine-based approaches to control malaria.

Previous honorees include Oleh Hornykiewicz of the Medical University of Vienna and the University of Toronto; Roger Nicoll of the University of California, San Francisco; and Solomon Snyder of the Johns Hopkins University School of Medicine in 2014 for research into neurotransmission and neurodegeneration.

David Botstein of Princeton University and Ronald Davis and David Hogness of Stanford University School of Medicine received the Alpert Prize in 2013 for contributions to the concepts and methods of creating a human genetic map.

Alain Carpentier of Hôpital Européen Georges-Pompidou in Paris and Robert Langer of MIT received the prize in 2011 for innovations in bioengineering.

Harald zur Hausen and Lutz Gissmann of the German Cancer Research Center received the prize in 2007 for their research on human papillomavirus (HPV) and cancer of the cervix. Hausen and others were later honored with the 2008 Nobel Prize in Physiology or Medicine.

The Warren Alpert Foundation

Each year the Warren Alpert Foundation receives 30 to 50 nominations for the Alpert Prize from scientific leaders worldwide. Prize recipients are selected by the foundation’s scientific advisory board, composed of distinguished biomedical scientists and chaired by the dean of Harvard Medical School.

Warren Alpert (1920-2007), a native of Chelsea, Massachusetts, established the Warren Alpert Foundation Prize in 1987 after reading about the development of a vaccine for hepatitis B. Alpert decided on the spot that he would like to reward such breakthroughs, so he picked up the phone and told the vaccine’s creator, Kenneth Murray of the University of Edinburgh, that he had won a prize. Alpert then set about creating the foundation.

To award subsequent prizes, Alpert asked Daniel Tosteson (1925-2009), then dean of Harvard Medical School, to convene a panel of experts to identify scientists from around the world whose research has had a direct impact on the treatment of disease.

The Warren Alpert Foundation does not solicit funds. It is a private philanthropic organization funded solely by the Warren Alpert Estate.

News

High-fidelity CRISPR

Improved gene-editing tool has no detectable off-target mutations 

A new version of the CRISPR-Cas9 nuclease appears to robustly abolish the unwanted, off-target DNA breaks that are a significant current limitation of the gene-editing technology. 

Harvard Medical School researchers at Massachusetts General Hospital describe in Nature how engineering the Cas9 enzyme to reduce nonspecific interactions with the target DNA may greatly expand applications of the CRISPR-Cas9 technology.

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“Our creation of a Cas9 variant that brings off-target effects to levels where we can no longer detect them, even with the most sensitive methods, provides a substantial advance for therapeutic applications in which you want to accurately hit your target without causing damage anywhere else in the genome,” said J. Keith Joung, HMS professor of pathology at Mass General and senior author of the Nature paper. 

“We envision that our high-fidelity variant will supplant the use of standard Cas9 for many research and therapeutic applications.”— J. Keith Joung

“Its impact will also be incredibly important for research applications because off-target effects can potentially confound the results of any experiment,” Joung added. “As a result, we envision that our high-fidelity variant will supplant the use of standard Cas9 for many research and therapeutic applications.”

The limitation

Used to create targeted DNA breaks at which genetic changes can be introduced, CRISPR-Cas9 nucleases combine a bacterial DNA-cutting enzyme called Cas9 with a short guide RNA sequence that can bind to the target DNA sequence. While easier to use than previous gene-editing tools, CRISPR-Cas9 nucleases have a well-characterized and significant limitation. 

As described in 2013 studies led by Joung and others, CRISPR-Cas9 nucleases can induce off-target DNA breaks at sites that resemble the on-target sequence. Subsequent investigations by Joung’s team and others have reduced but never completely and consistently eliminated these off-target effects.

Joung and his colleagues hypothesized that reducing interactions between Cas9 and the target DNA might more completely eliminate off-target effects while still retaining the desired on-target interaction. The team focused on the fact that certain portions of the Cas9 enzyme itself can interact with the backbone of the target DNA molecule. 

Pursuing an observation originally made by co-lead author Vikram Pattanayak, HMS clinical fellow in pathology at Mass General, the team altered four of these Cas9-mediated contacts by replacing the long amino acid side chains that bind to the DNA backbone with shorter ones unable to make those connections. 

“Our previous work suggested that Cas9 might bind to its intended target DNA site with more energy than it needs, enabling unwanted cleavage of imperfectly matched off-target sites,” said Pattanayak. “We reasoned that, by making substitutions at these four positions, we could remove some of that energy to eliminate off-target effects while still retaining full on-target activities.”

Testing the fix

Co-lead author Benjamin Kleinstiver, HMS research fellow in pathology at Mass General, and Michelle Prew, a research technician in Joung’s lab, then tested all 15 possible variants in which any combination of one, two, three or four of those amino acid side chains were altered. They found that one three-substitution and one four-substitution variant appeared to show the greatest promise in discriminating against mismatched target sites while retaining full on-target activities in human cells. 

The researchers then more fully characterized the four-substitution variant, which they called SpCas9-HF1; Sp for Streptococcus pyogenes bacteria, which is the source of this widely used Cas9, and HF for high fidelity. They found that this variant induced on-target effects comparable to those observed with the original unaltered SpCas9 when used with more than 85 percent of 37 different guide RNAs they tested.

Using GUIDE-Seq, a highly sensitive system Joung’s lab developed in 2014 to detect off-target CRISPR-Cas9 effects across the genome, the team found that, while nucleases combining unaltered SpCas9 with seven different guide RNAs induced as many as 25 off-target mutations, use of SpCas9-HF1 produced no detectable off-target effects with six of those guide RNAs and only one off-target site with the seventh. These results were further confirmed using targeted deep-sequencing experiments.

Joung’s team also found that SpCas9-HF1 could reduce off-target effects when targeting atypical DNA sites characterized by repeat sequences of one or two nucleotides—sites that are typically subject to many off-target mutations. They developed additional derivatives of SpCas9-HF1—called HF2, HF3 and HF4—which could eliminate the few residual off-target effects that persisted with the HF1 variant and a small number of guide RNAs.

Engineering new variants

“If SpCas9-HF1 using a certain guide RNA still produces a handful of off-target effects that are particularly difficult to eliminate, it may be possible to engineer new variants that get rid of even those effects,” said Joung. 

The researchers also showed that SpCas9-HF1, like its naturally occurring counterpart, could be combined with other useful alterations that extend its utility. Previous work from the Joung lab published last summer in Nature showed that introducing a series of amino acid substitutions could expand the targeting range of unaltered SpCas9.

In the current study, the authors show that introducing these same alterations into SpCas9-HF1 also extended the targeting range of the high-fidelity variant.

“These results show that these variants should be broadly useful to anyone currently using CRISPR-Cas9 technology,” said Kleinstiver. “They can easily be used in place of wild-type SpCas9 and provide a highly effective method for reducing off-target mutations to undetectable levels.”

Support for the study includes National Institutes of Health (NIH) Director’s Pioneer Award DP1 GM105378, NIH grants R01 GM107427 and R01 GM088040, the Jim and Ann Orr MGH Research Scholar Award, and the Natural Sciences and Engineering Research Council of Canada.

Adapted from a Mass General news release.

News

Hearts and Minds

Genetic underpinning found for shared risk of congenital heart and neurodevelopmental disease

Children with significant congenital heart disease have a far better chance of surviving today than in decades past, thanks to major advances in surgery. But some infants who recover from repairs to their hearts later show the effects of delays in brain development, including impairments to cognitive, language and social functioning. Such impairments can affect how well these children do in school and in the workplace; they can even diminish their overall quality of life.

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Epidemiological studies have given numbers to what doctors and families have long observed: The risk of neurodevelopmental delays is tenfold higher for children with moderate to severe congenital heart disease than for other children.

But why?

Over the years, those who study these phenomena have considered several possible reasons. Do the rigors of open-heart surgery so soon after birth play a role? Could heart defects limit nutrients and oxygen needed by the fetus? Or could spontaneous genetic mutations cause congenital problems that affect both the heart and the brain of a child?

Now, the “why” may have been answered by the efforts of the Pediatric Cardiovascular Genetics Consortium, led by a team of Harvard Medical School scientists. In a recent issue of Science the consortium reported exome sequence analyses of more than 1,200 children and their parents and showed that children with both congenital heart disease and neurodevelopmental delays share certain genetic mutations that thwart the normal development of both the heart and the brain.

“We’re homing in on a set of genes that have multiple different roles on multiple different tissues during development: heart tissue, brain tissue, other developing organs, limb tissue.” —Jason Homsy

Using a mathematical model created by co-authors Kaitlin Samocha and Mark Daly of the Analytical and Translational Genetics Unit at Massachusetts General Hospital, the team analyzed mutations in the protein-coding portion of the genomes of children with congenital heart disease that were not present in their parents’ genomes. They found that these children have more of these de novo mutations in genes that are highly expressed in the developing heart, compared to a control cohort of children without congenital heart disease. 

The de novo mutations were also found to be more frequent in children with congenital heart disease plus another birth defect, either neurodevelopmental delay or more-subtle abnormalities of finger or ear shape. These findings bolster the case for shared genetic causes of the cardiac and extra-cardiac abnormalities rather than surgeries or environmental factors.

“We’re homing in on a set of genes that have multiple different roles in multiple different tissues during development: heart tissue, brain tissue, other developing organs, limb tissue,” said Jason Homsy, an HMS LaDue Fellow who trained at Mass General and co-lead author of the Science paper. “Our study shows a common genetic link for the development of these diseases.”

Potential for early testing

According to Homsy and co-senior author Christine Seidman, the HMS Thomas W. Smith Professor of Genetics and Medicine at Brigham and Women’s Hospital and a Howard Hughes Medical Institute investigator, these findings could lead to early testing that would help identify newborns with congenital heart disease who are at high risk of neurodevelopmental difficulties.

“We can pretty clearly tell the parents of children with congenital heart disease what’s going to happen after the heart surgery, but there’s always a big question: Will my kid learn well in school?” Seidman said. “If we could identify children at high risk for neurodevelopmental delays, they could receive increased surveillance and earlier interventions than occur now.”  

The mutations primarily affected genes involved in three areas: morphogenesis, chromatin modification and transcriptional regulation. If any one of these processes is perturbed even slightly at a critical time in development, the heart is malformed; sometimes another developmental defect occurs, such as a missed connection in the brain.

'Master regulators'

“These genes are not just involved in shaping the heart,” Seidman said. “They are master regulators of organ development.”

One of the mutated genes is RBFOX2, which encodes a molecule that regulates RNA splicing. Although RBFOX2 has not been previously implicated in congenital heart disease, de novo mutations were identified in multiple affected children.

“There are still many unanswered questions, including why the same mutation can cause very different clinical manifestations,” Seidman said. Perhaps additional genetic variants in the multiple layers of transcriptional regulation allow compensation for some mutations but worsen the consequences of others. For now, Seidman said, knowing that a genetic mutation is present is different from knowing the outcome.

“It’s a long, long, long way down the road,” Seidman said, “but we’d like to believe that if you knew the steps by which these mutations perturbed the regulation of gene expression, there might even be ways to actually treat it.”

This work was supported by grants from the National Heart, Lung, and Blood Institute and the National Human Genome Research Institute of the National Institutes of Health, Howard Hughes Medical Institute, Simons Foundation for Autism Research, John S. LaDue Fellowship at HMS, Medical Scientist Training Program and National Research Science Award, Academy of Medical Sciences, British Heart Foundation, Wellcome Trust, Arthritis Research UK and the NIHR Cardiovascular Biomedical Research Unit at Royal Brompton and Harefield NHS Foundation Trust and Imperial College London, Leducq Foundation, Heart and Stroke Foundation of Ontario, Ted Rogers Centre for Heart Research, Kostin Family Innovation Fund, Aaron Stern Professorship at the University of Michigan, and Braylon’s Gift of Hope Fund.

News

On the Frontier of Genetic Technologies

Members of the Personal Genetics Education Project brief Congress on gene editing advances  

WASHINGTON, D.C.—Genetics is not your destiny, George Church told an overflow audience gathered in a Congressional briefing room. This was one of many head-turning statements he and two other speakers made about advances in the rapidly progressing field of genetic technologies.

“You can change it,” Church said about the genome. “Certainly you need to know it first.”

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In a lightning-quick 10 minutes, Church, the Robert Winthrop Professor of Genetics at Harvard Medical School, explained how gene editing can potentially eliminate malaria and allow transplants of pig organs into humans, among other innovations.

Change was the theme of a briefing designed to shed light on the frontiers of genetic technologies. Church was joined by Diana Bianchi, the Natalie V. Zucker Professor at Tufts University School of Medicine, and Jennifer Doudna, professor of molecular and cell biology and of chemistry at the University of California, Berkeley.

“This is a technology that seemed at first like science fiction: a technology for rewriting DNA itself. We can sequence DNA, we can find mutations. What if we had a way to fix those mutations? That technology is not science fiction; it is a reality.”— Jennifer Doudna, University of California, Berkeley

The Nov. 17 briefing was presented by the Personal Genetics Education Project (pgEd), a multifaceted effort based in the Department of Genetics at HMS. The mission of pgED is to educate individuals about advances in the field through school programs, libraries, religious institutions and other public forums, and to accelerate public awareness of genetics issues by advising the entertainment industry. It also seeks to engage lawmakers—the “eyes and ears of the nation”—in discussions.

pgEd takes no position on policy, preferring to educate from a neutral position so that its audience can make better-informed decisions.

Each speaker was passionate about the need to inform and educate the public about genetics and the power of new technologies to affect health and disease.

Bianchi is a leading medical geneticist in the burgeoning field of prenatal diagnosis. Doudna and her colleague Emmanuelle Charpentier, now at the Max Planck Institute for Infection Biology, are credited with translating an ancient mechanism in bacterial immunity into a powerful tool for editing genomes. Church optimized the gene-editing technology, now known by the acronym CRISPR, by developing what are known as gene drives, that is, molecular tools that can “drive” a DNA sequence to be inherited by all of an organism’s offspring. Jennifer Doudna. Image: John Boal

Doudna described the technology she and others have advanced as a genome-engineering revolution.

“This is a technology that seemed at first like science fiction; a technology for rewriting DNA itself,” she said. “We can sequence DNA, we can find mutations. What if we had a way to fix those mutations? That technology is not science fiction; it is a reality.”

The possible therapeutic applications are promising, she said, but delivery into cells and tissues remains a challenge.

Doudna also pointed out the fundamental difference between gene editing that affects only one person versus gene editing in the germ line that is passed on to future generations.

“When do we apply that? And for what kind of afflictions?” she asked. “I’m very interested myself in trying to stimulate public conversation over the implications of this technology.”

Bianchi, who both sees patients and conducts research in medical genetics, is already using genomic sequencing in her practice. Non-invasive prenatal testing takes advantage of the fact that small amounts of DNA fragments from the placenta circulate within the blood of pregnant women. Cell-free fetal DNA can be analyzed to screen for extra or missing copies of chromosomes that might indicate Down syndrome or other inherited syndromes.

Diana Bianchi. Image: John Boal

Since 2011, the DNA screening tests have been done on 2 million women. The DNA tests have both a high sensitivity and specificity and most important, Bianchi said, a higher positive predictive value than the current noninvasive screening tests that measure blood proteins and fetal ultrasound markers.

“Genomic testing has really changed prenatal medicine,” Bianchi said. “We think the future is linked to treatment.”

Bianchi’s lab is testing in mice whether administering therapy during pregnancy improves fetal brain development. Treatment in pregnant women could start as early as 12 weeks following a confirmed diagnosis of fetal Down syndrome and continue until delivery at 40 weeks.

If such a strategy works, it would give pregnant women three options to consider: continuing the pregnancy aware of a high risk for a genetic abnormality, terminating the pregnancy or treating the condition before birth.

Church explained how gene drives could make mosquitoes immune to malaria and therefore unable to transmit the disease to humans. 

“That would have a huge impact. Like smallpox, once it’s gone, it’s gone,” he said.

Gene editing could also eliminate the viral components of the pig genome, thereby making organ transplants into humans more feasible. A recent paper showed that 62 genes could be altered at once, demonstrating the power of the technology.

While such projects are tantalizing, concern is also growing among scientists, bioethicists, policymakers and others over how gene-editing technologies, such as CRISPR, will be used, the speakers said, underscoring the importance of public discussion.  

George Church. Image: John BoalThe briefing on Capitol Hill was sponsored by U.S. Rep. Louise Slaughter, D-N.Y., and Sen. Elizabeth Warren, D-Mass. More than 160 people, half of them Congressional staffers, heard where the science of genetic technologies is today, where it may be going and what concerns exist over its benefits and risks.

“It’s all about scalability,” said Ting Wu, HMS professor of genetics and pgEd’s director and co-founder. She hoped to send people out of the room astounded by the science and eager to spark discussion among policymakers, thus amplifying pgEd’s reach.

After the briefing, the speakers and the pgEd team met with the White House Office of Science and Technology, where they expressed hope that all segments of society could become part of the discussion.

“We think it is critical to engage across all communities, particularly the disenfranchised,” Marnie Gelbart, pgEd director of programs, said later. “This piece is as important as the technologies themselves, for safely and fairly integrating the technologies into society.”

The briefing was the fourth presented by pgEd. The first briefing highlighted the science of genomics, personalized medicine and genetic engineering, as well as ways to reach out to the public. The second focused on two topics: the role of genetics research in the unfolding Ebola outbreak in West Africa and the issues addressed by the Genetic Information Nondiscrimination Act, also known as GINA. The third centered on law enforcement and highlighted research on how microbial genomics is being used to improve health and increase public safety. For the fourth, pgEd wanted to bring attention to the excitement at the forefront of genetics and the questions being raised about how we as a society use these new technologies.

pgEd is supported by the HMS Department of Genetics and private funding from Sigma-Aldrich, Autodesk, Genentech, IDT (targeted specifically for GETed conferences and Map-Ed) and an anonymous donor.

News

Time for a New Definition

Researchers uncover diverse subtypes of serotonin-producing neurons

It used to be enough to call a serotonergic neuron a serotonergic neuron.

These brain cells make the neurotransmitter serotonin, which helps regulate mood, appetite, breathing rate, body temperature and more.

Recently, however, scientists have begun to learn that these neurons differ from one another—and that the differences likely matter in dysfunction and disease.

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Last year, a team led by Harvard Medical School genetics professor Susan Dymecki defined a subgroup of serotonergic neurons in mice by showing that those cells specifically, among all serotonergic neurons, were responsible for increasing the breathing rate when too much carbon dioxide builds up in the body.

Now, Dymecki and colleagues have taken a first stab at systematically characterizing serotonergic neurons at the molecular level and defining a full set of subtypes, again in mice.

The researchers report in Neuron that serotonergic neurons come in at least six major molecular subtypes defined by distinct expression patterns of hundreds of genes. In many cases, the subtypes modulate different behaviors in the body.

By conducting a cross-disciplinary series of experiments, the researchers found that the subtypes also vary in their developmental lineage, anatomical distribution, combinations of receptors on the cell surface and electrical firing properties.

“This work reveals how diverse serotonin neurons are at the molecular level, which may help to explain how, collectively, they are able to perform so many distinct functions,” said Benjamin Okaty, a postdoctoral researcher in the Dymecki lab and co-first author of the paper.

“To have the list of molecular players that make each of these subtypes different from one another gives us an important handle on learning more about what that cell type does and how we can manipulate only that subtype,” said Dymecki. “It holds enormous therapeutic potential.”

“This is an ancient neurotransmitter system that’s implicated in many different diseases, and it’s starting to be cracked open,” said Morgan Freret, a graduate student in the Dymecki lab and co-first author of the paper. “We can now ask questions in a more systematic way about which serotonergic cells and molecules are important in, for example, pain, sleep apnea or anxiety.”

Crucially, the team also showed that a serotonergic neuron’s gene expression and function depend not only on its location in the adult brain stem, but also on its cellular ancestor in the developing brain.

“Earlier work had shown that you could explore the relationship between a mature neuronal system and the different developmental lineages that gave rise to it, but we had no idea whether it was meaningful,” said Dymecki. “We show that the molecular phenotypes of these neurons track quite tightly to their developmental origin, with anatomy making some interesting contributions as well.”

While the work was done in mice, Dymecki is optimistic that it will be replicated in humans because the serotonergic neuronal system is in a highly conserved region of the brain, meaning it tends to remain consistent across vertebrate species.

Because of this, researchers can look for the same molecular signatures in human tissue and begin to tease apart whether particular subtypes of serotonergic neurons are involved in conditions such as sudden infant death syndrome (SIDS) or autism.

Such research could ultimately reveal previously unknown contributions of the serotonergic neuronal system to disease, inform the development of biomarkers or lead to more targeted therapies.

The team’s findings could also inform stem cell research. “Which subtype of serotonergic neuron are we getting when we use current stem cell protocols?” asked Dymecki. “Can we drive the development of different subtypes? Can we watch how gene expression patterns change over time during development for each subtype?”

Finally, the study provides an example of a highly integrative approach to understanding brain function at multiple scales, “linking genes and gene networks to the properties of single neurons and populations of neuron subtypes, all the way up to the level of animal behaviors,” said Okaty. “I think it’s a useful template going forward. Imagine what we’d learn by applying this approach to all the neurotransmitter systems in the brain.”

This research was supported by funding from the National Institutes of Health (R01 DA034022, P01 HD036379, T32 GM007753, R21 MH083613, R21 DA023643), the American SIDS Institute, a Harvard Stem Cell Institute seed grant, a NARSAD Distinguished Investigator Grant from the Brain and Behavior Foundation, and Harvard’s Blavatnik Biomedical Accelerator, which provides resources to develop early-stage biomedical technologies toward clinical applications. Harvard’s Office of Technology Development has filed a patent application on the technology.

News

MicroRNAs and Metabolism

Genetic study reveals four tiny RNA segments control cholesterol and lipid levels

Four tiny segments of RNA appear to play critical roles in controlling cholesterol and triglyceride metabolism. In their report in Nature Medicine, Harvard Medical School researchers at Massachusetts General Hospital describe how these microRNAs could reduce the expression of proteins playing key roles in generating beneficial HDL cholesterol, disposing of artery-clogging LDL cholesterol, controlling triglyceride levels and other risk factors for cardiovascular disease.

“While we and others have recently identified microRNAs that control cholesterol and fat metabolism and trafficking, no studies to date have systematically looked at all non-coding factors such as microRNAs in genetic studies of human diseases and other traits,” said Anders Näär, HMS professor of cell biology and corresponding author of the current study. 

“Using human genetic data from almost 190,000 individuals, we have linked 69 microRNAs to increased genetic risk for abnormal cholesterol and triglyceride levels, and showed that four of these act to control proteins we know are involved in those metabolic activities,” Näär said.

“We hope these findings will lead to new, more effective ways of treating or even preventing cardiovascular disease and other metabolic disorders.”— Anders Näär

Less than 2 percent of human DNA represents genes that code for the production of proteins. While it was originally hypothesized that the other 98 percent had no function—leading to the term “junk DNA”—it has now become apparent that these DNA sequences play essential roles in determining how, when and where protein-coding DNA is expressed. 

One such control mechanism is through single-stranded microRNAs, which block the expression of protein-coding genes by binding to messenger RNAs and preventing their translation into protein. In previous studies, Näär and his colleagues found that a microRNA called miR-33 suppresses production of beneficial HDL cholesterol and that antisense blocking of miR-33 increased HDL levels in an animal model.

The current study began with analysis of genome-wide association studies involving more than 188,000 people. The researchers identified 69 microRNAs located near gene variants previously associated with lipid abnormalities.

Using a tool that predicts the targets of microRNAs based on matches between their nucleotide sequences and those of protein-coding genes and a database of identified gene functions, the researchers arrived at four microRNAs that appear to control genes involved in cholesterol and triglyceride levels and in other metabolic functions, such as glucose metabolism.

Two of these—miR-128-1 and miR-148a—were found to control the expression of proteins essential to the regulation of cholesterol/lipid levels in cells and in animal models; miR-128-1 was also found to regulate fatty liver deposits, insulin signaling and maintenance of blood sugar levels. 

“We are following up these findings with studies to address whether antisense blocking of these microRNAs could decrease atherosclerosis, cardiovascular disease and inflammatory fatty liver diseases in animals,” Näär said. “We hope these findings will lead to new, more effective ways of treating or even preventing cardiovascular disease and other metabolic disorders.”

Support for the study includes National Institutes of Health grants R21DK084459, R01DK094184, R37DK048873, R01DK056626, K24DK078772, R01HL107953 and R01HL106063. 

Adapted from a Mass General news release.

News

Diverging Cancer Paths

Brain metastases harbor different genetic mutations from those in primary tumors

Brain metastases are a devastating complication of cancer, leading to the death of more than half of patients whose cancer spreads to the brain. A new study finds that while brain metastases share some genetic characteristics with the primary tumors from which they originated, they also carry unique genetic mutations.

The diverging evolutionary pathways of the metastatic and the primary tumors may change sensitivities to targeted therapy drugs, an international collaboration led by Harvard Medical School scientists at Massachusetts General Hospital report in Cancer Discovery.

“Our study demonstrates that while brain metastases and primary tumors share a common ancestry, they continue to evolve separately,” said Priscilla Brastianos, HMS instructor in medicine at Mass General and co-lead author of the paper.

“This is tremendously important, as we demonstrate that brain metastases may have clinically significant mutations that have not been detected either in the primary tumor biopsy or in metastases from other parts of the body,” she said. “We also showed that multiple brain metastases from the same patient share nearly all clinically significant mutations.”

Brain metastases commonly develop from melanoma, lung cancer or breast cancer and can appear despite the primary tumor’s being well controlled by drugs that target mutations driving its growth. Once brain metastases develop, patients usually die within a matter of months, and patients with brain metastases are typically excluded from most clinical trials.

“While brain metastases and primary tumors share a common ancestry, they continue to evolve separately.” —Priscilla Brastianos

In treating cancers known to be driven by targetable gene mutations, treatment planning is usually based on genetic analysis of tissue from the primary tumor. Because treatment of brain metastases often involves removal of the metastasis, samples of that tumor are often available for analysis. The current study was designed to investigate whether the genetic profiles of brain metastases are identical to those of the primary tumors.

The research team conducted whole-exome gene sequencing on three tissue samples—primary tumor, brain metastasis and normal tissue—from each of 86 patients with lung, breast or kidney cancers. The exome is the tiny portion of the genome that encodes proteins.

In each instance, while the investigators found that the primary tumor and the metastasis shared some mutations, the brain metastases had new mutations that were not related to those of the primary tumors. In four of the 86 patients, the brain metastases actually appeared to have originated from additional primary tumors.

The new mutations detected in the metastases often signaled potential sensitivity to targeted therapy drugs that would not have been effective against the primary tumors. Overall, more than half of the patients appeared to have clinically targetable new mutations in their brain metastases.

Analysis of multiple brain metastases samples from the same patient showed that nearly all of the significant mutations appeared in all of the brain metastases. In contrast, metastases from other parts of the body differed significantly from the brain metastases.

“It has been unclear whether brain metastases from well-controlled primary tumors develop because the chemotherapy drugs don’t cross the blood-brain barrier or because of different genetic mutations in the metastasis,” said Brastianos. “Our data suggest that genetic differences may contribute to the formation and treatment resistance of brain metastases.”

The clinical impact of directly targeting brain-metastasis specific mutations needs to be evaluated more fully, she said, and it is something the scientists are now investigating.

“We believe that routinely looking for clinically significant alterations in brain metastases may open the door to new therapeutic options for these patients,” Brastianos said.

Support for the study includes National Institutes of Health grants U54 HG003067, 5U24 CA143687 and U54 CA143798, and grants from the Brain Science Foundation, Susan G. Komen for the Cure, Terri Brodeur Breast Cancer Foundation, Conquer Cancer Foundation, the American Brain Tumor Association, Breast Cancer Research Foundation and the Mary Kay Foundation.

Adapted from a Mass General news release.

News

Stephen J. Elledge Receives Lasker Award

Geneticist recognized for discovering DNA repair pathway

Stephen J. Elledge, the Gregor Mendel Professor of Genetics and of Medicine at HMS, and professor of medicine at Brigham and Women’s Hospital, is a co-recipient, with Evelyn Witkin of Rutgers University, of the 2015 Albert Lasker Basic Medical Research Award.

The award, widely considered to be among the most respected in biomedicine, will be presented on Friday, Sept. 18, in New York City.

Elledge and Witkin are being honored for their seminal discoveries that have illuminated the DNA damage response, a cellular pathway that senses when DNA is altered and sets in a motion a series of responses to protect the cell. This pathway is critical to a better understanding of many diseases and conditions, such as cancer.

"Steve is an amazing scientist, mentor and colleague," said Jeffrey S. Flier, dean of Harvard Medical School.

"His insights into the basic mechanisms of the DNA damage response have profoundly enriched our understanding not only of the fundamental genetics of all cellular life, but also of how we conceptualize many diseases and conditions, especially cancer. This distinction is richly deserved, and I am delighted that Steve is being honored for this extraordinary body of work," Flier said.

"We are extremely proud of Steve, who is truly deserving of this recognition,” said Elizabeth G. Nabel, MD, president of Brigham and Women's Health Care.

“Courageous and insatiably inquisitive, he represents the best of Brigham and Women’s and our mission of driving innovation in basic science to improve human health. As a devoted mentor, Steve is deeply committed to guiding the careers of young investigators, ensuring that the next generation of scientists is filled with curious, passionate and talented researchers," she added.

Elledge often describes the process by which a cell duplicates itself as akin to the duplication of a small city. It is a vastly complex process that requires many levels of intricate coordination. Each cell contains a detailed blueprint for this entire process: DNA.

But not every duplication results in a perfect copy. That is because each time a cell makes a copy of itself, DNA is vulnerable to damage, not only from faulty cellular processes, but also from such entities as environmental chemicals. As DNA damage accumulates, it profoundly complicates a cell’s ability to make a faithful copy of itself. This can lead to serious illnesses, birth defects, cancer and other health problems.

Witkin discovered how bacteria respond to DNA damage, detailing the response to UV radiation. Elledge uncovered a DNA-damage response pathway that operates in more complex organisms, including humans.

Over the years, Elledge and his colleagues elucidated a signaling network that informs a cell when DNA sustains an injury.

Called the DNA damage response, this network senses the problem and sends a signal to the rest of the cell so it can properly repair itself, otherwise severe mutations can occur. As a result, this pathway helps keep the genome stable and suppresses adverse events such as tumor development.

When individuals are born with mutations in this pathway, they often have severe developmental defects. If the pathway is interfered with later in life, cancer can result.

In addition to the award in basic medical research, the Lasker Foundation is also presenting awards to individuals in clinical research and in public service.

According to Claire Pomeroy, president of the Albert and Mary Lasker Foundation, this year’s recipients “remind us all that investing in biological sciences and medical research is crucial for our future.”

Joseph L. Goldstein of the University of Texas Southwestern Medical Center and chair of the Lasker Medical Research Awards Jury, added, “The 2015 Lasker winners had bold ideas and pursued novel questions that they tested through fearless experimentation.”

News

Damage Control

Researchers connect pathway linked to Parkinson’s disease with genes mutated in ALS

When mitochondria—the cell’s power plant—are sick or damaged, they must be cleared away so the cell can survive. 

In the brain, this mitochondrial quality control pathway is so critical that neurodegenerative disease can result if bad mitochondria accumulate in neurons.

Now, Harvard Medical School researchers have mechanistically connected this pathway—which is already linked to Parkinson’s disease—with proteins that are mutated in amyotrophic lateral sclerosis, the motor-neuron disease also known as Lou Gehrig’s disease. Their findings are described in Molecular Cell.

In healthy cells, damaged mitochondria are broken down and disposed of by a process called selective autophagy, a term stemming from Greek roots that translates as “self-eating.” How the mitochondria get tagged for disposal has been the subject of research in many labs around the world in recent years.

Proteins linked to Parkinson’s

Mitochondrial damage triggers activation of two proteins—PARKIN and PINK1—that tag the mitochondria surface with chains of ubiquitin, molecules that signal the cell to get rid of the defective mitochondria. These proteins have been known for more than a decade to be faulty in early-onset familial forms of Parkinson’s disease, but only recently have their roles in mitochondrial quality control been elucidated.

Most recently, scientists have been studying how cells recognize these disposal signals and which proteins are involved further downstream in the disposal process.

Initial key insights into the downstream mechanism were suggested several years ago by the finding that the autophagy cargo receptor protein OPTN functions together with the TBK1 protein kinase in the removal of pathogenic bacteria from cells via autophagy. Interestingly, both OPTN and very recently TBK1 were also found to be mutated in ALS, but how these proteins contribute to this neurodegenerative disease has remained poorly understood.

Now scientists led by Wade Harper have mechanistically connected these two sets of proteins and have described a multistep mitochondrial quality control pathway. Upstream proteins—PARKIN and PINK1—function early in the mitochondrial disposal process by assembling ubiquitin chains on damaged mitochondria while the downstream proteins—OPTN and TBK1—bind to these ubiquitin chains to target the damaged mitochondria to the autophagy machinery.

Importantly, binding of the OPTN-TBK1 complex to these ubiquitin chains promotes TBK1 activation and further activation of OPTN’s ubiquitin-binding activity, establishing a self-reinforced feed-forward mechanism that is critical for the ultimate delivery of mitochondria to the autophagosome.

Connection to ALS

By using state-of-the-art quantitative mass spectrometry coupled with gene-editing and cell-imaging tools, the new study provides a detailed picture of how these two pathways work together and suggests how they may link the two neurodegenerative diseases.

“This is the first time we could mechanistically show how ubiquitination of damaged mitochondria promotes their clearance, and suggests that mitophagy defects potentially contribute to ALS as well as Parkinson’s disease,” said Harper, who is Bert and Natalie Vallee Professor of Molecular Pathology and chair of the HMS Department of Cell Biology.

Alban Ordureau, a postdoctoral fellow in the Harper lab and a co-author of the paper, had previously studied how PARKIN and PINK1 work together biochemically to specifically tag damaged mitochondria with ubiquitin.

“The story here focuses on how, once mitochondria are ubiquitinated, they are recognized by the cell and gotten rid of by proteins implicated in two distinct neurodegenerative diseases,” said Ordureau.

Jin-Mi Heo, postdoctoral fellow in the Harper lab and lead author of the paper, discovered that TBK1 becomes activated when mitochondria are damaged via ubiquitin chain binding by OPTN.

Stress and sensitivity

“If there is stress happening in the same cell in one pathway, that might cause defects in other pathways,” Heo said. “That could be why we see similar characteristics in different diseases with different origins.”

In Parkinson’s disease, neurons that make the neurotransmitter dopamine are defective while in ALS, the motor neurons do not perform as they should.

“There are some very complex relationships between different mutations that are going to lead to disease and which cell types they act in,” Harper said. “Depending on the cell type, you get one or the other disease.”

One idea is that individual types of neurons are sensitive to toxic proteins or toxic organelles—such as damaged mitochondria—in different ways. Motor neurons, for example, are much longer than most neurons in the brain, so they may be more sensitive to the accumulation of certain types of toxic proteins or organelles than other types of neurons, Harper said. The mutations that are present in the individual diseases may reflect sensitivities to different types of autophagic cargo; the result is motor neuron disease in ALS or dopaminergic disease in Parkinson’s.

“The surprising thing is that the Parkinson’s genes are functioning upstream of a pathway that’s mutated downstream in motor neuron disease,” Harper said. “So there is a genetic sensitivity within the pathway that must be different in different cells.”

It may turn out that this is a general mechanism that cells use to get rid of a variety of damaged material in different kinds of neurons.

“We’re starting to work that out,” Harper said.

This study was supported by National Institutes of Health grant R37 NS083524, Biogen and the Edward R. and Anne G. Lefler Center for the Study of Neurodegenerative Disorders at HMS.  Harper is a consultant for Millennium: the Takeda Oncology Company and Biogen.

News

Global Ambition

Cancer biology and therapeutics program debuts 

Harvard Medical School and the Qatar Biomedical Research Institute, with a commitment from the Shenzhen-HMS Initiative in International Education in China, are working together to make history with a joint global initiative designed to disseminate knowledge of cancer biology, prevention and treatment.

A new 12-month program aimed at international clinicians and scientists who specialize in cancer, Cancer Biology and Therapeutics will break new ground as the first HMS global program to combine basic and applied science in a blended-learning format, providing insights from leading researchers, oncologists and therapeutic developers both online and in person.

“This collaborative initiative will benefit Qatar Biomedical Research Institute, the Shenzhen-HMS Initiative in International Education and Harvard Medical School through the exchange of ideas and will develop a regional and global network of future leaders in cancer research and treatment,” said Ajay Singh, HMS associate dean for global education and continuing education.

HMS-CBT, as the program is known, highlights the global nature of the HMS mission, said George Demetri, co-director of the Harvard Ludwig Center, HMS professor of medicine at Dana-Farber Cancer Institute and a co-director of HMS-CBT.

The deadline for applications is Oct. 14.

“We are very focused on the terrific talent we’re pleased to have in the United States, but many brilliant people outside the United States are not able to come here for their training,” Demetri said.

“The more we can share the expanding body of knowledge about cancer biology and therapeutics with them, I think the better it will be for patients and for the field,” he said.

Combining the study of cancer biology with the study of cancer therapeutics is intentional and necessary, Demetri said.

“We have had an explosion in knowledge relating to cancer—the diversity of cancers, the fact that cancer is not one disease but probably thousands of different diseases that we can now parse very finely in the new world of precision cancer medicine,” he said.

“How do we develop therapeutics from that knowledge? How do we apply those therapeutics? That has so much depth and robustness we can address in this program,” Demetri added.

Emphasis on interactivity

Through a combination of three in-person, three-day workshops in cities on three continents—in Doha (Qatar), London and Boston—the program emphasizes interactivity between students and Harvard faculty and between groups of students who will organize themselves via internet-connected teams around projects and ideas.

The program, which begins Oct. 24 with a workshop in Doha, will be multi-faceted, reflecting both the evolving science of cancer and the interests of its students.  The first workshop will feature an introductory session on cancer biology, followed by sessions focused on links between infectious diseases, viruses and cancer.

The second workshop, starting March 18, 2016, in London, will focus on targeted therapies in cancer.

The third workshop and capstone event for the program, beginning Oct. 26, 2016, in Boston, will feature the latest developments in cancer immunotherapy, a dynamic field that is changing so rapidly that the organizers predict topics will evolve over the coming year.

Students will also be asked about what they are particularly interested in learning.

'Bespoke'

HMS-CBT program co-director Ed Harlow, the Virginia and D.K. Ludwig Professor of Cancer Research and Teaching in the HMS Department of Biological Chemistry and Molecular Pharmacology, is well known for Provocative Questions, a program he initiated in 2012 at the National Cancer Institute with former director Harold Varmus.  Harlow remains special assistant to the current NCI director.

Harlow hopes to spark the same impulse with students at the first HMS-CBT workshop by inviting them to ask questions for which there may not be one simple answer.

“This sounds interesting to me, this idea that an international group of scientists will be talking to one another about this project they’ve got to work on, and how they’ll do it across global time zones and cultures,” Harlow said.

Demetri calls this adaptable form of education “bespoke.” While a library of lectures from Harvard faculty is being assembled, webinars and interaction among students and with professors will supplement them, addressing the needs and interests of the students as they emerge. 

Peer-to-peer projects

The program directors are committed to interactivity, which they say will sprout at the workshops and flower during the year through peer-to-peer projects.

“The course will create opportunities for the students to network,” said Peter Howley, the Shattuck Professor of Pathological Anatomy in the HMS Department of Microbiology and Immunobiology and an HMS-CBT co-director.

“One could imagine wonderful and creative outcomes from such learning collaborations. The course faculty will give them assignments and will be around to help the students refine and work on them, but the actual learning is going to be among themselves,” he said.

In the process, the program directors will actively assess the course to learn what succeeds, and, along with the students, will work to build upon the experiences for future offerings of this course.

”We want to provide an educational experience that’s useful for all the participants,” Demetri said.

“It is really going to be an exciting year.”   

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