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News Article | August 26, 2016
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The genome-editing system known as CRISPR allows scientists to delete or replace any target gene in a living cell. MIT researchers have now added an extra layer of control over when and where this gene editing occurs, by making the system responsive to light. With the new system, gene editing takes place only when researchers shine ultraviolet light on the target cells. This kind of control could help scientists study in greater detail the timing of cellular and genetic events that influence embryonic development or disease progression. Eventually, it could also offer a more targeted way to turn off cancer-causing genes in tumor cells. “The advantage of adding switches of any kind is to give precise control over activation in space or time,” said Sangeeta Bhatia, the John and Dorothy Wilson Professor of Health Sciences and Electrical Engineering and Computer Science at MIT and a member of MIT’s Koch Institute for Integrative Cancer Research and its Institute for Medical Engineering and Science. Bhatia is the senior author of a paper describing the new technique in the journal Angewandte Chemie. The paper’s lead author is Piyush Jain, a postdoc in MIT’s Institute for Medical Engineering and Science. Before coming to MIT, Jain developed a way to use light to control a process called RNA interference, in which small strands of RNA are delivered to cells to temporarily block specific genes. “While he was here, CRISPR burst onto the scene and he got very excited about the prospect of using light to activate CRISPR in the same way,” Bhatia said. CRISPR relies on a gene-editing complex composed of a DNA-cutting enzyme called Cas9 and a short RNA strand that guides the enzyme to a specific area of the genome, directing Cas9 where to make its cut. When Cas9 and the guide RNA are delivered into cells, a specific cut is made in the genome; the cells’ DNA repair processes glue the cut back together but permanently delete a small portion of the gene, making it inoperable. In previous efforts to create light-sensitive CRISPR systems, researchers have altered the Cas9 enzyme so that it only begins cutting when exposed to certain wavelengths of light. The MIT team decided to take a different approach and make the binding of the RNA guide strand light-sensitive. For possible future applications in humans, it could be easier to deliver these modified RNA guide strands than to program the target cells to produce light-sensitive Cas9, Bhatia said. “You really don’t have to do anything different with the cargo you were planning to deliver except to add the light-activated protector,” she said. “It’s an attempt to make the system much more modular.” To make the RNA guide strands light-sensitive, the MIT team created “protectors” consisting of DNA sequences with light-cleavable bonds along their backbones. These DNA strands can be tailored to bind to different RNA guide sequences, forming a complex that prevents the guide strand from attaching to its target in the genome. When the researchers expose the target cells to light with a wavelength of 365 nanometers (in the ultraviolet range), the protector DNA breaks into several smaller segments and falls off the RNA, allowing the RNA to bind to its target gene and recruit Cas9 to cut it. In this study, the researchers demonstrated that they could use light to control editing of the gene for green fluorescent protein (GFP) and two genes for proteins normally found on cell surfaces and overexpressed in some cancers. “If this is really a generalizable scheme, then you should be able to design protector sequences against different target sequences,” Bhatia said. “We designed protectors against different genes and showed that they all could be light-activated in this way. And in a multiplexed experiment, when a mixed population of protectors was used, the only targets that were cleaved after light exposure were those being photo-protected.” This precise control over the timing of gene editing could help researchers study the timing of cellular events involved in disease progression, in hopes of determining the best time to intervene by turning off a gene. “CRISPR-Cas9 is a powerful technology that scientists can use to study how genes affect cell behavior,” said James Dahlman, an assistant professor of biomedical engineering at Georgia Tech, who was not involved in the research. “This important advance will enable precise control over those genetic changes. As a result, this work gives the scientific community a very useful tool to advance many gene editing studies.” Bhatia’s lab is also pursuing medical applications for this technique. One possibility is using it to turn off cancerous genes involved in skin cancer, which is a good target for this approach because the skin can be easily exposed to ultraviolet light. The team is also working on a “universal protector” that could be used with any RNA guide strand, eliminating the need to design a new one for each RNA sequence, and allowing it to inhibit CRISPR-Cas9 cleavage of many targets at once. The research was funded by the Ludwig Center for Molecular Oncology, the Marie-D. and Pierre Casimir-Lambert Fund, a Koch Institute Support Grant from the National Cancer Institute, and the Marble Center for Cancer Nanomedicine.

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After its emergency meeting on Zika virus two weeks ago, World Health Organization (WHO) Director-General Margaret Chan wrote in an official statement, “the Committee advised that the recent cluster of microcephaly cases and other neurological disorders reported in Brazil, following a similar cluster in French Polynesia in 2014, constitutes an ‘extraordinary event’ and a public health threat to other parts of the world.” Given these stakes, WHO declared Zika a “global public health emergency." The designation highlights immediate goals, such as “development of new diagnostics . . . prioritized to facilitate surveillance and control measures,” and longer-term measures, such as “appropriate research and development efforts . . . intensified for Zika virus vaccines, therapeutics and diagnostics.” MIT’s Institute of Medical Engineering and Science (IMES) is on the front lines of this research. Led by Professor Lee Gehrke, the MIT Gehrke Lab is at the vanguard of disease diagnostics. In recent years, lab members have developed a successful, noncross-reacting diagnostic test for Dengue virus, a close relative to Zika, as well as one for Ebola. “The Gehrke team is currently focused on developing sensitive, low-cost, rapid diagnostics to detect Zika, giving a result in 10 to 15 minutes, as well as on basic research needed to characterize Zika virus infections in advance of therapeutics and vaccine development,” says Gehrke, the Hermann von Helmholtz Professor in IMES and Professor of Microbiology and Immunobiology at Harvard Medical School. “Detection is needed from an epidemiological perspective . . . [and] to improve patient care. By detecting specific pathogens, a tailored treatment plan can be developed.” The threat is compounded by several factors, Gherke says: “Because there is little immunity against Zika virus infections in most parts of the world, Zika has the potential to spread anywhere that the mosquito vectors (Aedes aegypti and Aedes albopictus) are found. “Further, there are few available research reagents that can be used for scientific experimentation and study. Yet the development and characterization of reagents needed to detect Zika virus is underway at a rapid pace now, [and] we hope that the Zika test will be ready for field-testing in late spring or early summer 2016. Gehrke adds that his team has been indespensible in the work: “MIT graduate student Helena de Puig is developing nanoparticle surface chemistries to increase the sensitivity of the tests, and undergraduate Luis Mora is taking time from his work on the MIT Solar Car to precision-mold the cartridges that hold the device,” he says. Because Zika disease is often nonsymptomatic, several public health concerns are presently at play, Gehrke says. “There might be some danger from nonsymptomatic individuals serving as virus sources for mosquitoes, who could in turn infect other people,” he explains. “Once an individual has been infected, it’s reasonable to predict that they will have immunity, but no one knows the long term effects of Zika infections.” For this reason, Gehrke’s lab wants to provide health workers with a rapid test to detect who has Zika and does not, as well as who’s had it in the past. U.S. Centers for Disease Control Director Tom Friedan wrote for CNN: “Diagnosing prior infection with Zika is much more challenging. . . . This is a priority, and we are working to do in weeks what would usually take months or years.” “Identifying individuals who have been infected in the past is done by identifying anti-Zika IgM and IgG antibodies,” Gehrke says. “Advanced technology is available for examining the portfolio of pathogens that have infected an individual over time. However, as a practical matter with readily available reagents, the IgG and IgM tests can be non-specific. Our Dengue Fever device can identify IgG and IgM in patient serum, and we will develop the same capacity for Zika. Crossover interactions with nonspecific antigens are, however, a consideration.” Since reporting outbreaks is crucial to public health, technology is also being implemented to make diagnostics into messengers. “The detection technology is being linked to low-cost communication and data technologies that will analyze the test results and transmit them to personal care physicians and public health officials,” Gehrke explains. “[IMES researcher] Jose Gomez Marquez is adapting a 5-dollar Raspberry Pi single-board computer to invent a very low-cost reader that will quantify the rapid test data. More research is needed to determine how that data can be transmitted and stored to protect privacy while improving communication with physicians and public health officials.” Additionally, the Gehrke Lab wants to help officials to “develop a plan for a ‘smart’ mosquito trap that identifies mosquito species as they enter and transmits the data to a home base. We are also exploring technologies to screen mosquitoes directly for the presence of viruses, in an effort to provide advance warning of an impending epidemic.” Ultimately, the sudden, intensive focus on Zika could help the fight against other viruses, as well. Gehrke’s own lab is focused on detecting multiple RNA viruses, zeroing in on processes from innate immune signaling to gene expression. “Because the genomes and proteins of the flaviviruses (including Zika) are related, it is quite possible that studying Zika and comparing its replication strategies and pathogenesis to other viruses will be very informative,” Gehrke says. Daniel Anderson, an associate professor of chemical engineering and member of IMES, is working on a new type of vaccine: a customized, on-hand, single-dose RNA nanoparticle vaccine — that may have potential to fight the spread of Zika virus. “We have developed a nanoparticle vaccine that can be rapidly tuned for different diseases, including viruses. The speed of development allows us to make new vaccines in only seven days, allowing the potential to deal with sudden outbreaks, like Zika,” Anderson says. “Using a single injection, these nanoparticles deliver RNA, carrying instructions that ultimately help train the patient’s immune system to fight off infections.” The team at the Anderson Lab has successfully developed customized vaccines for all three classes of National Institute of Allergy and Infectious Disease’s “priority pathogens,” including Ebola virus (class A), H1N1 Influenza (class B), and microorganism parasite Toxoplasma gondii (class C), generating fully protective immunity in animals. “We have already started using the same process for Zika,” says postdoc Omar F. Khan, who works closely with Anderson at the Koch Institute for Integrative Cancer Research at MIT. “It’s possible that some of the antigens found in Zika are also present in other arboviruses [viruses caused by mosquito or tick bites], which broadens the vaccine’s applicability.” In fact, by designing the synthetic nanoparticle to “simultaneously deliver many different and unique RNAs,” the Anderson Lab’s vaccine innovation has the ability to target multiple diseases, reducing the burden on health care workers addressing large populations. “These can be multiple antigens from the same virus or different antigens from other viruses. We call this multiplexing,” Khan says. “In this way, it’s possible to make a pan-arbovirus vaccine that can simultaneously train the immune system to target [related viruses]. We are already doing this type of multiplexing for filoviruses. Our pan-filovirus nanoparticle vaccine is designed to simultaneously target Zaire Ebola, Sudan Ebola, and Marburg virus, and is currently being validated by our collaborators at the United States Army Medical Research Institute for Infectious Diseases.” The speed, cost, and on-demand, local production potential of a nanoparticle vaccine for Zika — or Zika combined with other arboviruses — makes it particularly compelling. “Once the unique Zika antigens have been identified by virology experts, it will take us about seven days to generate the Zika-specific RNA instructions and build the nanoparticle vaccines,” Anderson says. “We are working with labs and government agencies that study the Zika virus to validate the potential in preventing this disease.” The nanoparticle vaccine is, according to Anderson’s presentations, the “first and only nonviral replicon delivery system that has achieved protective immunity in lethal exposure experiments . . . [with] no side effects [and] no risk of anti-vector immunity” — in other words, no risk of being attacked by the patient’s immune system. And based on its success preventing other viruses, influenza, parasites, and even some cancers in animals, it offers promising potential as a formidable foe to Zika in the future, if clinical trials are prioritized.

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Scientists at MIT, Massachusetts General Hospital, Living Proof, and Olivo Labs have developed a new material that can temporarily protect and tighten skin, and smooth wrinkles. With further development, it could also be used to deliver drugs to help treat skin conditions such as eczema and other types of dermatitis. The material, a silicone-based polymer that could be applied on the skin as a thin, imperceptible coating, mimics the mechanical and elastic properties of healthy, youthful skin. In tests with human subjects, the researchers found that the material was able to reshape “eye bags” under the lower eyelids and also enhance skin hydration. This type of “second skin” could also be adapted to provide long-lasting ultraviolet protection, the researchers say. “It’s an invisible layer that can provide a barrier, provide cosmetic improvement, and potentially deliver a drug locally to the area that’s being treated. Those three things together could really make it ideal for use in humans,” says Daniel Anderson, an associate professor in MIT’s Department of Chemical Engineering and a member of MIT’s Koch Institute for Integrative Cancer Research and Institute for Medical Engineering and Science (IMES). Anderson is one of the authors of a paper describing the polymer in the May 9 online issue of Nature Materials. Robert Langer, the David H. Koch Institute Professor at MIT and a member of the Koch Institute, is the paper’s senior author, and the paper’s lead author is Betty Yu SM ’98, ScD ’02, former vice president at Living Proof. Langer and Anderson are co-founders of Living Proof and Olivo Labs, and Yu earned her master’s and doctorate at MIT. As skin ages, it becomes less firm and less elastic — problems that can be exacerbated by sun exposure. This impairs skin’s ability to protect against extreme temperatures, toxins, microorganisms, radiation, and injury. About 10 years ago, the research team set out to develop a protective coating that could restore the properties of healthy skin, for both medical and cosmetic applications. “We started thinking about how we might be able to control the properties of skin by coating it with polymers that would impart beneficial effects,” Anderson says. “We also wanted it to be invisible and comfortable.” The researchers created a library of more than 100 possible polymers, all of which contained a chemical structure known as siloxane — a chain of alternating atoms of silicon and oxygen. These polymers can be assembled into a network arrangement known as a cross-linked polymer layer (XPL). The researchers then tested the materials in search of one that would best mimic the appearance, strength, and elasticity of healthy skin. “It has to have the right optical properties, otherwise it won’t look good, and it has to have the right mechanical properties, otherwise it won’t have the right strength and it won’t perform correctly,” Langer says. The best-performing material has elastic properties very similar to those of skin. In laboratory tests, it easily returned to its original state after being stretched more than 250 percent (natural skin can be elongated about 180 percent). In laboratory tests, the novel XPL’s elasticity was much better than that of two other types of wound dressings now used on skin — silicone gel sheets and polyurethane films. “Creating a material that behaves like skin is very difficult,” says Barbara Gilchrest, a dermatologist at MGH and an author of the paper. “Many people have tried to do this, and the materials that have been available up until this have not had the properties of being flexible, comfortable, nonirritating, and able to conform to the movement of the skin and return to its original shape.” The XPL is currently delivered in a two-step process. First, polysiloxane components are applied to the skin, followed by a platinum catalyst that induces the polymer to form a strong cross-linked film that remains on the skin for up to 24 hours. This catalyst has to be added after the polymer is applied because after this step the material becomes too stiff to spread. Both layers are applied as creams or ointments, and once spread onto the skin, XPL becomes essentially invisible. The researchers performed several studies in humans to test the material’s safety and effectiveness. In one study, the XPL was applied to the under-eye area where “eye bags” often form as skin ages. These eye bags are caused by protrusion of the fat pad underlying the skin of the lower lid. When the material was applied, it applied a steady compressive force that tightened the skin, an effect that lasted for about 24 hours. In another study, the XPL was applied to forearm skin to test its elasticity. When the XPL-treated skin was distended with a suction cup, it returned to its original position faster than untreated skin. The researchers also tested the material’s ability to prevent water loss from dry skin. Two hours after application, skin treated with the novel XPL suffered much less water loss than skin treated with a high-end commercial moisturizer. Skin coated with petrolatum was as effective as XPL in tests done two hours after treatment, but after 24 hours, skin treated with XPL had retained much more water. None of the study participants reported any irritation from wearing XPL. “I think it has great potential for both cosmetic and noncosmetic applications, especially if you could incorporate antimicrobial agents or medications,” says Thahn Nga Tran, a dermatologist and instructor at Harvard Medical School, who was not involved in the research. Living Proof has spun out the XPL technology to Olivo Laboratories, LLC, a new startup formed to focus on the further development of the XPL technology. Initially, Olivo’s team will focus on medical applications of the technology for treating skin conditions such as dermatitis. Other authors of the paper include Fernanda Sakamoto and Rox Anderson of MGH; Soo-Young Kang of Living Proof; Morgan Pilkenton and Alpesh Patel, formerly of Living Proof; and Ariya Akthakul, Nithin Ramadurai, and Amir Nashat ScD ’03, of Olivo Laboratories.

This technique, which can track changes in gene expression as cells differentiate, could be particularly useful for studying how stem cells or immune cells mature. It could also shed light on how cancer develops. "Existing methods allow for snapshot measurements of single-cell gene expression, which can provide very in-depth information. What this new approach offers is the ability to track cells over multiple generations and put this information in the context of a cell's lineal history," says Robert Kimmerling, a graduate student in biological engineering and the lead author of a paper describing the technique in the Jan. 6 issue of Nature Communications. The paper's senior authors are Scott Manalis, the Andrew and Erna Viterbi Professor of Biological Engineering and a member of MIT's Koch Institute for Integrative Cancer Research, and Alex Shalek, the Hermann L.F. von Helmholtz Career Development Assistant Professor of Health Sciences and Technology, an assistant professor of chemistry, and a member of MIT's Institute for Medical Engineering and Science. The new method incorporates a recently developed technology called single-cell RNA-seq, which sequences the messenger RNA from a single cell. These RNAs, known collectively as the transcriptome, reveal which genes are being actively transcribed (that is, copied into messenger RNA instructions for building proteins) inside a cell at a given point in time. This helps scientists understand, for example, what makes a skin cell so different from a heart cell even though the cells share the same DNA. "Scientists have well established methods for resolving diverse subsets of a population, but one thing that's not very well worked out is how this diversity is generated. That's the key question we were targeting: how a single founding cell gives rise to very diverse progeny," Kimmerling says. To try to answer that question, the researchers designed a microfluidic device that traps first an individual cell and then all of its descendants. The device has several connected channels, each of which has a trap that can capture a single cell. After the initial cell divides, its daughter cells flow further along the device and get trapped in the next channel. The researchers showed that they can capture up to five generations of cells this way and keep track of their relationships. To get the cells off the chip, the researchers temporarily reverse the direction of the fluid flowing across the chip, allowing them to remove the cells one at a time to perform single-cell RNA-seq. In this study, the researchers captured and sequenced immune cells called T cells. These cells are on constant alert in the body, and when they encounter a cell infected with a virus or bacterium, they leap into action, creating two distinct populations—effector T cells, which seek and destroy infected cells, and memory T cells, which retain a memory of the encounter and circulate in the body indefinitely in case of a subsequent encounter. "A single founding cell can give rise to both effector and memory cell subtypes, but how that diversity is generated isn't very clear," Kimmerling says. The researchers analyzed RNA from recently activated T cells and two subsequent generations. When comparing genes with functions related to T cell activation and differentiation, they found that "sister" cells produced from the same division event are much more similar in their gene expression profiles than two unrelated cells. They also found that "cousin" cells, which have the same "grandmother," are more similar than unrelated cells, which suggests unique, family-specific transcriptional profiles for single T cells. The researchers hope that future studies with this device could help to resolve the long-standing debate over how T cells differentiate into effector cells and memory cells. One theory is that the distinction occurs as early as the first T cell division following activation, while a competing theory suggests that the distinction happens later on, as a result of changes in the cells' microenvironment. To address this question, the researchers believe they would need to analyze the development of T cells taken from a mouse that had been exposed to a foreign pathogen, which would provide a useful model of T cell activation in response to infection. The new device could also be used to link RNA transcriptome information with other cell traits, the researchers say. "It opens up possibilities that have never been there before," Manalis says. "We can further annotate single-cell transcriptome data by applying this strategy to our existing devices for measuring mass, growth rate, density, or deformability." In this study, the researchers also discovered that they could use their new technique to learn which genes are expressed at certain points during the cell division cycle. Because they trap each cell and have a record of when it last divided, they can directly link the "age" of each cell to its transcriptome. They identified a set of about 300 genes that correspond most with time since division (a proxy for cell cycle progression), and found that most of those genes were involved in cell division. Therefore, by measuring the levels of those 300 genes in similar cells, scientists should be able to estimate the ages of those cells. The researchers also found that a leukemia cell line, which proliferates continuously, has a different set of genes that appear to be driving cell division. "In the future, this approach may be able to provide insight into unique transcriptional regulators of cell cycle progression in various cancer models," Kimmerling says. Explore further: New method for analysing RNA sequence data identifies new subtypes of cells

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New cancer drugs allow doctors to tailor treatment based on the genetic profile of a patient's tumor. However, these drugs don't work at all in some patients, and they lose their effectiveness in others. A new study from MIT and Massachusetts General Hospital reveals why a certain class of these drugs, known as kinase inhibitors, doesn't always halt tumor growth. The researchers found that while kinase inhibitors successfully shut down their targets, they also provoke cells to turn on a backup system that can take over for the one knocked out by the drug. The team also showed that disrupting both systems with a combination of drugs yields much better results, in a study of mice. "We've discovered a previously unappreciated mechanism involved in resistance to targeted therapeutics," says Douglas Lauffenburger, the Ford Professor of Bioengineering and head of MIT's Department of Biological Engineering. "Its presence appears to be associated with poor response to some kinase inhibitors in clinical patients. And we've demonstrated that in mice adding a drug against this resistance mechanism allows the original targeted drug to work when otherwise it wouldn't." Lauffenburger, who is also an affiliate member of MIT's Koch Institute for Integrative Cancer Research, is the senior author of the study, which appears in the March 16, 2016 online edition of Cancer Discovery. The lead authors are Miles Miller, a former MIT graduate student who is now a postdoc at Harvard Medical School, and Madeleine Oudin, a Koch Institute postdoc. Kinase inhibitors, frequently used against breast, ovarian, and other cancers, work by disrupting cell signaling pathways that stimulate cells to grow, proliferate, or become invasive. Doctors usually prescribe them based on whether a patient's tumor is overexpressing a cancer-driving protein such as epithelial growth factor receptor (EGFR). However, these drugs can fail even in tumors where they should work. About half of these failures are caused by genetic mutations that allow cancer cells to evade the drug's actions, but the rest are unexplained, Lauffenburger says. Based on their previous studies of endometriosis (when uterine tissue grows into surrounding organs), Lauffenburger and his colleagues suspected there could be a backup pathway helping cancer cells to sidestep the effects of kinase inhibitors. In those studies, the researchers found that invasive endometrial cells become "addicted" to a certain growth signal, and that this pathway actually shuts off other growth pathways. Drugs that shut down the primary pathway can have the unintended effect of activating those backup systems. The MIT team wondered if the same thing might be happening in cancer cells. They focused on melanoma and triple-negative breast cancer, two very aggressive forms of cancer that are often driven by EGFR ligands (molecules that activate the receptor), which help the cancer cells to become motile and invasive. They found that when EGFR ligands bind to receptors on the cancer cell surface, they not only trigger a cascade of cellular reactions that promotes invasiveness but also activate a positive feedback loop: Enzymes called proteases release EGFR ligands from the cell surface so they can bind to even more receptors, strengthening the pro-invasion signal. The researchers found that those proteases also chop off receptors that initiate other pro-invasion pathways. Essentially, the cancer cells become addicted to the EGFR-driven pathway and shut off competing pathways because they don't need them, Lauffenburger says. "The cells have the capability for other inputs, but if they're already signaling through one, they're perfectly happy to shut down the rest," he says. Consequently, when doctors give a kinase inhibitor that shuts off the EGFR pathway, it also shuts off the proteases, allowing the backup pathways, which are no longer being suppressed, to take over. The researchers also showed that these cleaved receptor proteins can be detected in blood samples from patients, and that the protein levels correlate with how well EGFR inhibitors work for individual patients. High levels of cleaved proteins mean that there is a lot of potential for the backup system to kick in, and kinase inhibitors will not be effective. However, if these protein levels are low, that suggests the backup system is not very strong in that patient's tumor. "The discovery seems to identify those patients who will go on to receive long-term clinical benefit versus those whose tumors will quickly adapt and circumvent treatment, by virtue of a blood-based test that can be performed at baseline or within days of initiating treatment," says Keith Flaherty, an author of the paper and director of developmental therapeutics at the MGH Cancer Center, who hopes to begin performing this kind of test in patients. The study also suggests that patients whose tumors have a strong backup system could benefit from receiving an EGFR inhibitor plus a drug that shuts down the secondary pathway. One candidate is a type of drug known as an AXL inhibitor, which is now in clinical trials. In mouse studies, the MIT team found that treating tumors with that combination of drugs was much more effective than giving either one alone.

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