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Sharper Image

Sharper Image

Researchers capture ultrasharp images of multiple cell components at once

by DAN FERBER
Mon, 2014-02-03

Ten-color super-resolution images (artificially arranged into one composite) of synthetic DNA nanostructures displaying patterns that resemble the numerals “0” through "9,” produced by Exchange-PAINT using only a single dye and a single laser source. Image: Johannes B. Woehrstein/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.

 

This ultrasharp Exchange-PAINT image simultaneously spots microtubules (green), mitochondria (purple), Golgi apparatus (red), and peroxisomes (yellow) from a single human cell. Image: Maier Avendano/Wyss Institute

 

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.

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Sharpening CRISPR-Cas's Aim

Sharpening CRISPR-Cas’s Aim

Shortening guide RNAs markedly improves gene-editing tool

By SUE McGREEVEY
Tue, 2014-01-28

Image: NIH's National Human Genome Research Institute.

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.

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'Barcode' profiling

'Barcode' profiling

New technology enables analysis of hundreds of tumor marker proteins at once

By SUE McGREEVEY and YVONNE FISHER-JEFFES
Tue, 2014-01-28

Image: iStock

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.

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Cancer Driver

Cancer Driver

Rare brain tumor carries BRAF mutation linked to other cancers

By SUE McGREEVEY
Mon, 2014-01-13

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.

CT scan of a craniopharyngioma. Image: Wikimedia Commons

“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.

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Cervical Cancer Clues

Cervical Cancer Clues

Genomic study points to potential therapeutics, highlights HPV

By VERONICA MEADE-KELLY
Thu, 2014-01-02

Squamous cell carcinoma in the cervix. Image: National Cancer Institute

Researchers from the Boston area, Mexico and Norway have completed a comprehensive genomic analysis of cervical cancer in two patient populations. The study identified recurrent genetic mutations not previously found in cervical cancer, including at least one for which targeted treatments have been approved for other forms of cancer. The findings also shed light on the role human papillomavirus (HPV) plays in the development of cervical cancer.

The study, which appears online in Nature, addresses a public health concern of global significance. Cervical cancer is the second most common cancer in women and is responsible for approximately 10 percent of cancer deaths in women—particularly in developing countries where screening methods are not readily accessible. Almost all cases of the disease are caused by exposure to HPV and it is expected that vaccination efforts targeting HPV will decrease cervical cancer cases over time. In the meantime, however, the disease remains a significant threat to women’s health.

“Cancer is a disease that affects the whole world, and one question always arises: Is a given cancer type similar or different across populations?” explained Matthew Meyerson, one of the paper’s co-senior authors. Meyerson is an HMS professor of pathology and medical oncology at Dana-Farber Cancer Institute and a senior associate member of the Broad Institute. “While we don’t have the complete answer yet in this case, what we are seeing is that, in two different populations, the causes of cervical cancer are similar and, fundamentally, in both cases it comes down to HPV-genome interaction.”

To investigate the genomic underpinnings of the disease, the team performed whole-exome sequencing, which examines the genetic code in the protein-coding regions of the genome, on samples from 115 cervical cancer patients from Norway and Mexico. In some cases, the researchers also conducted whole-genome sequencing (analyzing the genetic code across the entire genome) or transcriptome sequencing (focusing on gene expression). In each case, the researchers compared genomic data derived from cervical cancer tumors with genomic data from healthy tissue from the same individual to determine what may have gone wrong—or mutated—in the genome to allow the cancer to develop. The mutations identified in tumors but not in healthy tissues from the same individuals are referred to as somatic mutations.

The study benefited from the international collaboration of scientists from research institutes across the globe and was made possible by the Slim Initiative for Genomic Medicine in the Americas (SIGMA), which promotes the study of genomic medicine in the service of global health.

“Low- and middle-income countries suffer the largest burden of cancer in the world,” said co-author Jorge Melendez of the National Institute of Genomic Medicine in Mexico City. “Nevertheless, only 5 percent of all the global resources dedicated to this group of diseases are allocated to them. Initiatives that promote joint efforts with developing countries will help to advance not only the knowledge of the shared and distinct biological aspects of cancer diseases, but also highlight local action items to impact public health.”

The cooperation of teams from the U.S., Mexico and Norway was essential in order to sequence samples from a diverse pool of cervical cancer patients.

“Without this sort of international collaboration, the genomic view of a disease can be limited. By analyzing genomic data from diverse populations, we can discover patterns of disease progression in context of the full range of human genetic variation,” said co-senior author Helga Salvesen. Salvesen, a professor of clinical medicine at the University of Bergen, Norway, was a visiting scientist at the Broad Institute when the study was conducted. 

The study identified 13 mutations that occurred frequently enough across the samples to be considered significant in cervical cancer. Eight of these mutations had not been linked to the disease previously, and two had not previously been seen in any cancer type.

Among the most notable findings were somatic point mutations in the gene ERBB2, which was found in a small but significant subset of the tumors. Mutations in this gene, which is also known as HER-2, had not been previously linked to cervical cancer, but it is a known oncogene common in breast cancer. Treatments exist that target the gene.

“This suggests that a subset of cervical cancer patients could be candidates for clinical trials involving ERBB2 inhibitors, which are available and FDA approved,” explained Akinyemi Ojesina, an HMS postdoctoral fellow in Meyerson’s lab at the Broad Institute and Dana-Farber. Ojesina served as a co-first author of the paper along with Lee Lichtenstein of the Broad’s Cancer Genome Analysis Group. “It is an exciting finding that could be translatable to the clinic.”

The team also identified a novel mutation in the gene MAPK1. MAPK1 is one of the final steps in the MAP kinase signaling pathway—a network of interconnected genes that play a role in cell growth regulation. Mutations in other genes in the pathway have been known to drive cancer, but this is the first time that MAPK1 itself has been found to be mutated. The finding opens up the possibility that MAPK1, like other genes in the MAP kinase signaling pathway, may be a viable therapeutic target.

Another key finding was the prevalence of mutations in genes affecting the immune system, in particular those in the gene HLA-A, which helps the body distinguish its own proteins from foreign invaders. Mutations in HLA-A were previously found to drive squamous cell lung cancer. In this study, another gene in the same complex—HLA-B—was found to be commonly mutated in cervical squamous cell carcinoma. This suggests that disruptions to the immune system may play a bigger role in cancer progression than was previously realized.

Finally, transcriptome sequencing, which allowed the team to analyze gene expression—how and when genes are activated across the genome—enabled the researchers to learn more about how HPV is driving cervical cancer.

It has long been known that exposure to HPV is a primary risk factor for cervical cancer. Once an individual is exposed, the immune system often clears out the infection, but in cases in which the virus lingers, it can integrate itself into the human genome. This study looked at where in the genome HPV inserted itself. The scientists found that HPV integration sites were associated with higher levels of gene expression and were often amplified, resulting in many copies of those sections of the genome. This connection between HPV integration and gene expression suggests that the virus may be driving cancer by promoting and elevating the activity of mutated genes.

“Our findings further elucidate the key role HPV is playing in the development of cervical cancer, which in turn emphasizes the importance of combating the disease by vaccinating against HPV,” Meyerson said.

In addition to the evidence supporting vaccination as a means of prevention, the researchers say the study bears important clinical implications for targeted therapeutics.

“In metastatic cervical cancer, more effective systemic therapy is urgently needed,” Salvesen explained. “So far, our knowledge regarding genetic alterations as potential targets for therapeutics has been limited, and no targeted therapeutics are yet in routine clinical use. The present study—in particular the findings related to ERBB2—thus represents a unique and comprehensive new tool to guide clinical trial design in the future.”

“The outstanding findings of our successful collaborative international research is giving us, here in Mexico, very powerful arguments in favor of the benefit of speeding up the adoption of molecular methods for screening HPV, followed by colposcopy to detect cancer at its earliest phases when it is curable; not to mention the possibility of new targeted therapies that our discoveries open up,” said Hugo Barrera, a professor of biochemistry and molecular medicine at the School of Medicine and University Hospital of the University of Nuevo Leon in Monterrey, Mexico, who, with Melendez, coordinated the Mexican team.

“It is hoped that, as we combine vaccination strategies and novel targeted therapies, we will be better able to combat the scourge that is cervical cancer,” Ojesina added.

This work was conducted as part of the Slim Initiative for Genomic Medicine in the Americas, a project funded by the Carlos Slim Foundation through the Health Institute. This work was also partially supported by the Rebecca Ridley Kry Fellowship of the Damon Runyon Cancer Research Foundation; MMRF Research Fellow Award; Helse Vest, Research Council of Norway, Norwegian Cancer Society and Harald Andersens legat; CONACyT grant SALUD-2008-C01-87625 and UANL PAICyT grant CS1038-1; and CONACyT grant 161619.

Adapted from a Broad Institute news release.

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A New, Reversible, Cause of Aging

A New—and Reversible—Cause of Aging

A naturally produced compound rewinds aspects of age-related demise in mice

By DAVID CAMERON
Thu, 2013-12-19

Mitochondria, organelles on the right, interact with the cell's nucleus to ensure a healthy, functioning cell. Image by Ana GomesResearchers have discovered a cause of aging in mammals that may be reversible.

The essence of this finding is a series of molecular events that enable communication inside cells between the nucleus and mitochondria. As communication breaks down, aging accelerates. By administering a molecule naturally produced by the human body, scientists restored the communication network in older mice. Subsequent tissue samples showed key biological hallmarks that were comparable to those of much younger animals.

“The aging process we discovered is like a married couple—when they are young, they communicate well, but over time, living in close quarters for many years, communication breaks down,” said Harvard Medical School Professor of Genetics David Sinclair, senior author on the study. “And just like with a couple, restoring communication solved the problem.”

This study was a joint project between Harvard Medical School, the National Institute on Aging, and the University of New South Wales, Sydney, Australia, where Sinclair also holds a position.

The findings are published Dec. 19 in Cell.

Communication breakdown

Mitochondria are often referred to as the cell's "powerhouse," generating chemical energy to carry out essential biological functions. These self-contained organelles, which live inside our cells and house their own small genomes, have long been identified as key biological players in aging. As they become increasingly dysfunctional overtime, many age-related conditions such as Alzheimer’s disease and diabetes gradually set in.

Researchers have generally been skeptical of the idea that aging can be reversed, due mainly to the prevailing theory that age-related ills are the result of mutations in mitochondrial DNA—and mutations cannot be reversed.

Sinclair and his group have been studying the fundamental science of aging—which is broadly defined as the gradual decline in function with time—for many years, primarily focusing on a group of genes called sirtuins. Previous studies from his lab showed that one of these genes, SIRT1, was activated by the compound resveratrol, which is found in grapes, red wine and certain nuts.

Sirt1 protein, red, circles the cell's chromosomes, blue. Image by Ana GomesAna Gomes, a postdoctoral scientist in the Sinclair lab, had been studying mice in which this SIRT1 gene had been removed. While they accurately predicted that these mice would show signs of aging, including mitochondrial dysfunction, the researchers were surprised to find that most mitochondrial proteins coming from the cell’s nucleus were at normal levels; only those encoded by the mitochondrial genome were reduced.

“This was at odds with what the literature suggested,” said Gomes.

As Gomes and her colleagues investigated potential causes for this, they discovered an intricate cascade of events that begins with a chemical called NAD and concludes with a key molecule that shuttles information and coordinates activities between the cell’s nuclear genome and the mitochondrial genome. Cells stay healthy as long as coordination between the genomes remains fluid. SIRT1’s role is intermediary, akin to a security guard; it assures that a meddlesome molecule called HIF-1 does not interfere with communication.

For reasons still unclear, as we age, levels of the initial chemical NAD decline. Without sufficient NAD, SIRT1 loses its ability to keep tabs on HIF-1. Levels of HIF-1 escalate and begin wreaking havoc on the otherwise smooth cross-genome communication. Over time, the research team found, this loss of communication reduces the cell's ability to make energy, and signs of aging and disease become apparent.

“This particular component of the aging process had never before been described,” said Gomes.

While the breakdown of this process causes a rapid decline in mitochondrial function, other signs of aging take longer to occur. Gomes found that by administering an endogenous compound that cells transform into NAD, she could repair the broken network and rapidly restore communication and mitochondrial function. If the compound was given early enough—prior to excessive mutation accumulation—within days, some aspects of the aging process could be reversed.

When Sirt1 loses its ability to monitor HIF-1, communication between mitochondria and the nucleus breaks down, and aging accelerates. Image by Ana Gomes

Cancer connection

Examining muscle from two-year-old mice that had been given the NAD-producing compound for just one week, the researchers looked for indicators of insulin resistance, inflammation and muscle wasting. In all three instances, tissue from the mice resembled that of six-month-old mice. In human years, this would be like a 60-year-old converting to a 20-year-old in these specific areas.

One particularly important aspect of this finding involvesHIF-1. More than just an intrusive molecule that foils communication, HIF-1 normally switches on when the body is deprived of oxygen. Otherwise, it remains silent. Cancer, however, is known to activate and hijack HIF-1. Researchers have been investigating the precise role HIF-1 plays in cancer growth.

“It’s certainly significant to find that a molecule that switches on in many cancers also switches on during aging,” said Gomes. “We're starting to see now that the physiology of cancer is in certain ways similar to the physiology of aging. Perhaps this can explain why the greatest risk of cancer is age.”

“There’s clearly much more work to be done here, but if these results stand, then certain aspects of aging may be reversible if caught early,” said Sinclair.

The researchers are now looking at the longer-term outcomes of the NAD-producing compound in mice and how it affects the mouse as a whole. They are also exploring whether the compound can be used to safely treat rare mitochondrial diseases or more common diseases such as Type 1 and Type 2 diabetes. Longer term, Sinclair plans to test if the compound will give mice a healthier, longer life.

The Sinclair lab is funded by the National Institute on Aging (NIA/NIH), the Glenn Foundation for Medical Research, the Juvenile Diabetes Research Foundation, the United Mitochondrial Disease Foundation and a gift from the Schulak family.

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Silencing Sudden Death

Silencing Sudden Death

Study targets genetics of hypertrophic cardiomyopathy

By STEPHANIE DUTCHEN
Thu, 2013-10-03

The heart of a mouse with untreated hypertrophic cardiomyopathy (left) has a thicker muscle wall and more scarring (blue) than one treated with RNAi (right). Image: Seidman lab

Hypertrophic cardiomyopathy (HCM)—a disease in which cardiac muscle thickens, weakening the heart—can be prevented from developing for several months in mice by reducing production of a mutant protein, according to a new study by researchers at Harvard Medical School.

The work takes a first step toward being able to treat or prevent the leading cause of sudden death in athletes and sudden heart-related death in people under 30 in the United States.

“There’s really no treatment for HCM right now. You can treat symptoms like chest pain or an arrhythmia, but that’s not getting at the fundamental problem,” said Christine Seidman, the Thomas W. Smith Professor of Medicine and Genetics at HMS and Brigham and Women's Hospital, a Howard Hughes Medical Investigator and senior author of the study. “While the application of this strategy is in the very early stages, it shows considerable promise.”

The results were published in Science on Oct. 3.

An estimated 1 in 500 Americans has HCM. Although many of them never develop symptoms, for others the disease can be severe or fatal.

More than 1,000 different mutations that can cause HCM have been identified across about 10 genes that make heart muscle proteins. People with HCM have one “good” copy and one “bad” copy of one of those genes.

Studying one of the mutations that causes particularly severe disease, Christine Seidman and Jonathan Seidman, Henrietta B. and Frederick H. Bugher Foundation Professor of Genetics at HMS, worked with research fellow Jianming Jiang and instructor Hiroko Wakimoto to target the analogous “bad” gene in mice while leaving the “good” gene alone.

The researchers created an RNA interference (RNAi) tool designed to home in on the single HCM-causing mutation and stop it from making its harmful protein. They packaged the RNAi inside a virus (a common RNAi delivery technique) and injected it into lab mice engineered to develop HCM. They compared the results to two untreated groups of mice: one with the same HCM mutation, and one without.

By suppressing the “bad” gene, the RNAi was able to reduce production of the mutant protein by about 28 percent. That was enough to prevent development of HCM manifestations—including ventricular wall overgrowth, cell disorganization and fibrosis (scarring)—for about six months, or one-quarter of the mice's lifespans.

"For all intents and purposes, the heart looked normal," said Christine Seidman. "Wonderfully, boringly normal."

The treatment successfully targeted heart cells in the mice without affecting other organs. Although it did not reverse any existing HCM damage, Jonathan Seidman noted that halting the progress of HCM would be a significant advance in itself.

"If somebody already had a certain amount of wall thickness and you prevent it from worsening, that would be a step forward to limit progressive symptoms and development of heart failure," he said.

In addition to its potential for informing HCM treatment in humans down the road, the initial findings could be relevant for a related genetic condition called dilated cardiomyopathy, where the heart becomes baggy and thin-walled and contracts too little instead of too much.

The researchers now plan to investigate whether they can continue to delay HCM in mice with booster shots, reverse disease damage or reduce HCM-related arrhythmias. They would like to study a larger animal model as well as explore whether younger mice respond better to therapy than older mice and if interventions aimed at specific areas of the heart could be as effective as treating the whole heart.

The team also intends to explore whether a collection of about 10 RNAis could be engineered to target common genetic variants that are tightly linked to HCM mutations, instead of having to develop 1,000 mutation-specific RNAis.

The Seidmans are founders of and own shares in MyoKardia, a biotechnology company developing small molecules that target the sarcomere (the cellular structure that contracts in heart muscle) for treatment of inherited cardiomyopathy.

This work was supported by grants from the National Institutes of Health/National Heart, Lung, and Blood Institute (U01HL09166 and R01HL084553) and the Howard Hughes Medical Institute.

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Glowing Proteins: More Than a Marker

Glowing Proteins: More Than a Marker

Researchers transform fluorescent proteins into a scaffold for manipulating genes

By ELIZABETH COONEY
Thu, 2013-08-15

GFP (green) can be used not only as a fluorescent reporter for labeling cells, but also as a scaffold to control synthetic biological activities, such as gene expression (red). Image: Jonathan Tang, with contributions by Tim Cherry.

Jonathan Tang had a problem. A graduate student studying neural circuitry in the retina, he wanted to do more than identify fluorescent cells that send signals to the brain. He sought to understand how these specialized cells called bipolar neurons develop and function in the eye’s retina. More than “Here they are,” he hoped to say, “Here’s what they do.”

Frustrated by the lack of available tools, he created his own. Tang, with Constance Cepko, HMS Bullard Professor of Genetics and Neuroscience, and other collaborators, transformed green fluorescent protein (GFP), a glowing biomarker borrowed from jellyfish, into a scaffold that can bring together protein fragments to enable gene manipulation, or other useful activities.

Their method, described Aug. 15 in Cell, enables scientists to probe how cells function not only in the retina, but also in other tissues. The tool also works with the promising technology called optogenetics, in which scientists use light to control individual cells.

To solve his problem, Tang took a step back to look more closely at GFP. The green glow produced by this jellyfish protein has become a workhorse of science, literally illuminating pathways and processes in lab dishes and living animal models since its Nobel Prize-winning discovery in 1961 and application in 1994. With it, scientists can observe molecular biology occurring in more than 1,500 transgenic GFP strains in mice and other organisms, many of which label unique populations of cell types.

An invaluable resource, GFP is nonetheless limited to tagging cells. If scientists want to control gene activity, particularly in the mouse, they turn to another workhorse molecule, Cre recombinase, an enzyme that can shuffle DNA and is primarily used in the mouse. Not nearly as many mouse strains have been made to express Cre as have GFP, so for Tang to learn not just where bipolar cells are in the retina, but also how they develop and connect to other cell types, additional transgenic mice would need to be generated and characterized.

Tang decided not to wait. After all, he had a PhD to complete.

“Jonathan is the star of the story,” Cepko said. “We would sit and talk about how we had all these GFP lines we couldn't use to derive functional information. He came up with this idea that worked fantastically well, namely, to use the GFP to control biological activity specifically in cells already tagged with GFP.”

To get there, Tang focused not so much on the green fluorescence in GFP as on its possible uses as a protein. By itself GFP doesn’t alter cell function—an asset as a biomarker—but perhaps it could be combined with other proteins that affect gene activity. In that sense, GFP and its partners could act as both biomarker and synthetic system for controlling genes.

Searching for proteins that would bind to GFP, Tang discovered that a group of scientists in Germany had identified molecules that could bind to GFP. Derived from camel antibodies prized for their simple structure and high affinity, these proteins retained their ability to bind to GFP after they were introduced into a cell. Tang realized that this property could be exploited to create an array of tools that could enable the GFP-binding proteins to do much more. He constructed a series of chimeric, or fusion, proteins, fusing protein domains that can control transcription to these GFP-binding proteins. When two such fusion proteins that can bind simultaneously to GFP are introduced into a cell, GFP brings them together, triggering the designed co-dependent activity of the fusion proteins.

The scientists tested their tool in the retina, using it to switch on a fluorescent reporter gene in one experiment and to knock out a gene in another. As a boost to those who study neural function, they could trigger expression of channelrhodopsin, a protein that allows neural circuits to be controlled with light, thereby linking expression of GFP to the powerful tools of optogenetics. 

The tool worked in tissue culture as well as in living mice and zebrafish, another animal model favored by investigators.

“We can now use this as a way to access genetically defined cell types to see what’s essential for development, behavior, neural circuit processing—or whatever one wants to study,” said Tang.

“We can use these methods to probe function and development in the retina, but whatever your tissue is, you should be able to use these methods to study it,” Cepko said. “You don’t have to make a transgenic mouse or even have a GFP strain. You may be able to find an endogenous protein, or maybe even an RNA, that’s specific to your interest and do exactly what we did to make our tool.”

The study authors were supported by the Leonard and Isabelle Goldenson Research Fellowship, a European Research Council grant, a Swiss-Hungarian grant, TREATRUSH, SEEBETTER and OPTONEURO grants from the European Union and the Howard Hughes Medical Institute.
 

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New Project in IBD Genetics

New Project in IBD Genetics

Broad Institute and Mass General announce major sequencing initiative in inflammatory bowel disease

Fri, 2013-08-02

Image: iStock

The Broad Institute and Massachusetts General Hospital are launching a new initiative to perform large-scale exome sequencing in inflammatory bowel disease (IBD), a diagnosis—including Crohn’s disease and ulcerative colitis—that faces considerable unmet therapeutic need.

The recent emergence of rapid, efficient genome-sequencing technologies and the compelling evidence for the role of genetics in these disorders have motivated the founding of a collaborative sequencing effort between the two institutions with ties to Harvard Medical School. The endeavor will be geared toward the discovery of high-impact genetic variants influencing IBD risk that can serve as guides to future therapeutic development and diagnostic tools.

The initiative will be directed by experienced IBD researchers from Mass General and the Broad Institute, but it also aims to develop an exome-sequencing network that will allow researchers to partner and collaborate worldwide. Two large pilot exome-sequencing projects are being launched immediately as part of this initiative based on established genetic study designs that heighten the discovery of rare, high-impact risk and protective variants. The exome is the small portion of the genome that translates DNA into proteins.

Early-onset pediatric IBD

A major focus of the work will surround full-exome sequencing of the earliest-onset childhood IBD cases. It has long been recognized in many diseases that penetrant genetic risk factors—mutations in people who have symptoms—are much more likely to be found in cases with unusually early onset. The Broad Institute has completed more than 50,000 exomes in the past two years. Its technical expertise in generating and processing such sequencing data, along with population-level genetic variation patterns from these experiments, will provide a foundation for obtaining the best outcomes in the interpretation of these cases. Samples with IBD onset before 6 years of age—and in some cases going up to age 10—collected by IBD researchers around the world are welcome for inclusion in this study.

Adult-onset IBD case-control study

Genetic mapping has provided dramatic insights into IBD pathogenesis in recent years, but a substantial amount of heritability—in particular the role of strong-acting rare mutations—is yet to be elucidated. To deliver those insights, IBD cases with an unusually low burden of known genetic risk factors (particularly emphasizing those with positive family history, or from isolated or enriched populations, or both) will be contrasted with population controls that have an extremely high burden of known IBD risk factors, enhancing discovery of critical protective variants that may provide the best clues for therapeutic development. This project will also be initiated as an international collaboration, particularly among researchers with sample sets with Immunochip genotyping that will enable the immediate genetic identification of these enriched target populations. Immunochip is a tool that samples 200,000 sites in the genome previously tied to autoimmune and inflammatory diseases.

The National Human Genome Research Institute Large-Scale Sequencing Program will support pilot work for this project in the next 12 months, with all generated data made publicly available to the research community through standard National Institutes of Health databases.

“Genetics offers a peek into the pathways that protect against—or predispose to—the development of IBD,” said Ramnik Xavier, HMS professor of medicine and chief of gastroenterology at Mass General and a senior associate member at the Broad Institute. “Contributing results to public resources will allow the data to be integrated by researchers from a variety of disciplines. In the current research environment, important advances in biology are often made by finding innovative ways to analyze ‘big data,’ and I believe this open-access exome-sequencing project will provide an example of how publicly available data can inspire significant progress in the field of IBD.”

“This project will enable IBD genetics to take further leaps forward toward a clear and therapeutically actionable understanding of the molecular causes of disease,” said Mark Daly, HMS associate professor of medicine and chief of the Analytic and Translational Genetics Unit at Mass General and a senior associate member at the Broad Institute. “It is extremely exciting to be able to launch this transformative initiative as an international collaboration with rapid and open data sharing.”

Researchers who are interested in collaborating on this initiative may contact Daly or Xavier for more information.

Adapted from a joint Mass General and Broad Institute news release.

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Cholera's Evolution

Cholera’s Evolution

Study identifies genomic regions associated with cholera susceptibility in Bangladesh

By SUE McGREEVEY
Tue, 2013-07-16

People in the Ganges River Delta of Bangladesh have been affected by cholera for centuries. Image: NASA World Wind screenshot via Wikimedia Commons

An international research team has used a novel approach to identify genetic factors that appear to influence susceptibility to cholera, a disease that affects from 3 to 5 million people each year and causes more than 100,000 deaths. The findings indicate the importance of pathways involved in regulating water loss in intestinal cells and highlight the innate immune system’s key role in the body’s response to the bacteria that causes cholera.

The work involved investigators from Harvard Medical School and Massachusetts General Hospital; the Broad Institute; and the International Center for Diarrhoeal Disease Research, Bangladesh.

“We sought to understand cholera by studying the genetics of a population that has been affected by the disease for centuries: people in the Ganges River Delta of Bangladesh,” said Regina LaRocque, an assistant professor of medicine at Mass General and a co-senior author of the report published in Science Translational Medicine. “Our findings are just a first step, but they demonstrate how combining ancient history with the current impact of an infectious disease can be a powerful way of identifying human genes that are important to disease outcome.”

People contract cholera by consuming water or food contaminated with the bacteria Vibrio cholerae, which releases a toxic protein upon reaching the small intestine. This toxin binds to the intestinal surface, causing severe diarrhea and sometimes death from dehydration. Cholera or cholera-like illnesses have been reported in the Ganges Delta for centuries, and most recent global outbreaks of the disease originated in that region.

A potential fingerprint of cholera’s genetic impact could be the relative rarity in the region of people with blood type O, which confers an increased risk of severe cholera symptoms. The persistence of cholera in the Ganges Delta would be expected to exert an evolutionary force on the population, since individuals with gene variants that reduce their susceptibility to the disease would be more likely to survive and pass those variants along to their children.

To search for genomic regions that affect cholera susceptibility, the team employed a new two-step approach. The first step, developed by the Broad team, used a method called Composite of Multiple Signals (CMS) to scan the genomes of 126 individuals from the Ganges Delta for patterns that signal a long-term increase in the prevalence of particular DNA segments, indicating the effects of natural selection. That scan identified 305 regions under selective pressure, many of which are involved in two important biologic functions. One is regulation of the passage of water through intestinal cells via structures called potassium channels and the other is a signaling pathway involved in both the innate immune system and the maintenance of the intestinal lining.

The second step directly tested the potential impact of these selected regions on cholera susceptibility by comparing the genomes of 105 cholera patients from the region with the genomes of 167 individuals who did not contract the disease, despite being exposed to it in their homes. That comparison found that the genomic region most strongly associated with cholera susceptibility in this population was one that the CMS scan indicated was under strong selection pressure. Genes in this region relate to an innate immune signaling pathway. LaRocque’s team had previously shown this pathway to be activated by exposure to cholera toxin, and the current study identified the potential involvement of several additional genes in that pathway

“Understanding the basic biology of a disease is fundamental to making clinically relevant advances in treatment,” said LaRocque. “Our laboratory is now working on further studies of the innate immune response to cholera, and we believe this work will be highly relevant to developing improved vaccines.”

Support for the study includes National Institutes of Health grants TW007409, AI058935, AI079198 and NIH Innovator Award DP2-OD006514-01; grants from the Howard Hughes Medical Institute, the American Cancer Society and the Packard Foundation; and an MGH Claflin Distinguished Scholar Award.

Adapted from a Mass General news release.

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