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Researchers create synthetic cells to isolate genetic circuits

Anne Trafton |

Synthetic biology allows scientists to design genetic circuits that can be placed in cells, giving them new functions such as producing drugs or other useful molecules. However, as these circuits become more complex, the genetic components can interfere with each other, making it difficult to achieve more complicated functions.

MIT researchers have now demonstrated that these circuits can be isolated within individual synthetic “cells,” preventing them from disrupting each other. The researchers can also control communication between these cells, allowing for circuits or their products to be combined at specific times.

“It’s a way of having the power of multicomponent genetic cascades, along with the ability to build walls between them so they won’t have cross-talk. They won’t interfere with each other in the way they would if they were all put into a single cell or into a beaker,” says Edward Boyden, an associate professor of biological engineering and brain and cognitive sciences at MIT. Boyden is also a member of MIT’s Media Lab and McGovern Institute for Brain Research, and an HHMI-Simons Faculty Scholar.

This approach could allow researchers to design circuits that manufacture complex products or act as sensors that respond to changes in their environment, among other applications.

Boyden is the senior author of a paper describing this technique in the Nov. 14 issue of Nature Chemistry. The paper’s lead authors are former MIT postdoc Kate Adamala, who is now an assistant professor at the University of Minnesota, and former MIT grad student Daniel Martin-Alarcon. Katriona Guthrie-Honea, a former MIT research assistant, is also an author of the paper.

Circuit control

The MIT team encapsulated their genetic circuits in droplets known as liposomes, which have a fatty membrane similar to cell membranes. These synthetic cells are not alive but are equipped with much of the cellular machinery necessary to read DNA and manufacture proteins.

By segregating circuits within their own liposomes, the researchers are able to create separate circuit subroutines that could not run in the same container at the same time, but can run in parallel to each other, communicating in controlled ways. This approach also allows scientists to repurpose the same genetic tools, including genes and transcription factors (proteins that turn genes on or off), to do different tasks within a network.

“If you separate circuits into two different liposomes, you could have one tool doing one job in one liposome, and the same tool doing a different job in the other liposome,” Martin-Alarcon says. “It expands the number of things that you can do with the same building blocks.”

This approach also enables communication between circuits from different types of organisms, such as bacteria and mammals.

As a demonstration, the researchers created a circuit that uses bacterial genetic parts to respond to a molecule known as theophylline, a drug similar to caffeine. When this molecule is present, it triggers another molecule known as doxycycline to leave the liposome and enter another set of liposomes containing a mammalian genetic circuit. In those liposomes, doxycycline activates a genetic cascade that produces luciferase, a protein that generates light.

Using a modified version of this approach, scientists could create circuits that work together to produce biological therapeutics such as antibodies, after sensing a particular molecule emitted by a brain cell or other cell.

“If you think of the bacterial circuit as encoding a computer program, and the mammalian circuit is encoding the factory, you could combine the computer code of the bacterial circuit and the factory of the mammalian circuit into a unique hybrid system,” Boyden says.

The researchers also designed liposomes that can fuse with each other in a controlled way. To do that, they programmed the cells with proteins called SNAREs, which insert themselves into the cell membrane. There, they bind to corresponding SNAREs found on surfaces of other liposomes, causing the synthetic cells to fuse. The timing of this fusion can be controlled to bring together liposomes that produce different molecules. When the cells fuse, these molecules are combined to generate a final product.

More modularity

The researchers believe this approach could be used for nearly any application that synthetic biologists are already working on. It could also allow scientists to pursue potentially useful applications that have been tried before but abandoned because the genetic circuits interfered with each other too much.

“The way that we wrote this paper was not oriented toward just one application,” Boyden says. “The basic question is: Can you make these circuits more modular? If you have everything mishmashed together in the cell, but you find out that the circuits are incompatible or toxic, then putting walls between those reactions and giving them the ability to communicate with each other could be very useful.”

Vincent Noireaux, an associate professor of physics at the University of Minnesota, described the MIT approach as “a rather novel method to learn how biological systems work.”

“Using cell-free expression has several advantages: Technically the work is reduced to cloning (nowadays fast and easy), we can link information processing to biological function like living cells do, and we work in isolation with no other gene expression occurring in the background,” says Noireaux, who was not involved in the research.

Another possible application for this approach is to help scientists explore how the earliest cells may have evolved billions of years ago. By engineering simple circuits into liposomes, researchers could study how cells might have evolved the ability to sense their environment, respond to stimuli, and reproduce.

“This system can be used to model the behavior and properties of the earliest organisms on Earth, as well as help establish the physical boundaries of Earth-type life for the search of life elsewhere in the solar system and beyond,” Adamala says.

Artist Lecture: Ursula von Rydingsvard

Anne Trafton |

On October 28, 2016, the McGovern Institute held a dedication ceremony for SCIENTIA, a monumental bronze sculpture located at the entrance to the McGovern Institute at 550 Main Street in Cambridge, MA. SCIENTIA, by Ursula von Rydingsvard, is a gift from Lore Harp McGovern and represents the 52nd piece of public art on the MIT campus. In this video, von Rydingsvard explains the creative process involved in making SCIENTIA.

SCIENTIA

Anne Trafton |

The newest addition to MIT’s Public Art Collection is on permanent display at the entrance to the McGovern Institute for Brain Research. “SCIENTIA,” a monumental bronze sculpture by Ursula von Rydingsvard is a gift from Lore Harp McGovern and represents the 52nd piece of public art on campus. The sculpture was dedicated in a public ceremony on Friday, Oct. 28 at 5 p.m., followed by an artist talk. Photos of the sculpture and the dedication may be found below.

SCIENTIA

Anne Trafton |

SCIENTIA, 2016 by Itinerant Pictures

Documentary video on the making of SCIENTIA, a sculpture by Ursula von Rydingsvard located at the entrance to MIT’s McGovern Institute. The monumental sculpture was commissioned by Lore Harp McGovern for the McGovern Institute for Brain Research and the public art collection of the Massachusetts Institute of Technology.

McGovern Institute 2016 Halloween Party

Anne Trafton |

And the winner of this year’s costume contest (by popular vote) goes to Ruth Rosenholtz for her depiction of presidential candidate Donald Trump! See below for a full gallery of images from our annual Halloween party.

Monumental new sculpture commissioned for MIT’s McGovern Institute

by Julie Pryor |

The newest addition to MIT’s Public Art Collection is now on permanent display at the McGovern Institute for Brain Research at 550 Main Street in Kendall Square, Cambridge, Massachusetts. “SCIENTIA,” a monumental bronze sculpture by Ursula von Rydingsvard is a gift from Lore Harp McGovern and represents the 52nd piece of public art on campus. The new work will be dedicated in a public ceremony on Friday, Oct. 28 at 5 p.m., followed by a free artist talk at 6 p.m. in the Singleton Auditorium (Room 46-3002).

“’SCIENTIA’ represents that art and science are not separate entities,” says Lore Harp McGovern, co-founder of the McGovern Institute and a member of the Council for the Arts at MIT. “Art defines our humanity, advances our curiosity, and forces us to ask big questions — questions the McGovern Institute for Brain Research is trying to answer. ‘SCIENTIA’ invites you in.”

Von Rydingsvard’s “SCIENTIA” is among her most ambitious sculptures to date, at approximately 24 feet tall and over 17,000 pounds. In creating the work, the artist first produced a wood model in her studio using 4×4-inch cedar beams milled for the construction industry.  Using circular saws and a range of cutting tools, she sliced, marked, and shaped the wood elements, then stacked them to create layers that were glued and screwed into place. The full-scale wood model was then transported to Polich Tallix Fine Art Foundry (founded by Richard Polich SM ’65), where the majority of the sculpture was sand cast while the delicate filigree sections were cast using the lost-wax technique. Von Rydingsvard patinated the bronze surface by hand with chemicals and a blow torch.

For over 30 years, von Rydingsvard has been making monumental sculptures that reveal the trace of the human hand and resemble objects and environments that echo the artist’s family heritage in pre-industrial Poland. The artist’s childhood was marked by the strain of living in eight different refugee camps over the course of five years. Her earliest recollections — of displacement and subsistence through humble means — infuse her work with emotional potency. Von Rydingsvard has built towering cedar structures, creating intricate networks of individual beams, shaped by sharp and lyrical cuts and fused together to form rich, dynamic surfaces. While abstract at its core, von Rydingsvard’s work takes visual cues from the landscape, the human body, and utilitarian objects — such as the artist’s collection of household vessels — and demonstrates an interest in the point where the human-made meets nature.

“Ursula von Rydingsvard’s commissioned piece for the McGovern is a fantastic addition to MIT’s great public art collection,” List Visual Arts Center Director Paul C. Ha says. “This powerful sculpture will inspire many and will be one of the signature pieces in our collection. We’re grateful for Ms. McGovern’s thoughtfulness and her generosity in helping us acquire this magnificent piece for MIT.”

View the SCIENTIA photo gallery >>

A new player in appetite control

Anne Trafton |

MIT neuroscientists have discovered that brain cells called glial cells play a critical role in controlling appetite and feeding behavior. In a study of mice, the researchers found that activating these cells stimulates overeating, and that when the cells are suppressed, appetite is also suppressed.

The findings could offer scientists a new target for developing drugs against obesity and other appetite-related disorders, the researchers say. The study is also the latest in recent years to implicate glial cells in important brain functions. Until about 10 years ago, glial cells were believed to play more of a supporting role for neurons.

“In the last few years, abnormal glial cell activities have been strongly implicated in neurodegenerative disorders. There is more and more evidence to point to the importance of glial cells in modulating neuronal function and in mediating brain disorders,” says Guoping Feng, the James W. and Patricia Poitras Professor of Neuroscience. Feng is also a member of MIT’s McGovern Institute for Brain Research and the Stanley Center for Psychiatric Research at the Broad Institute.

Feng is one of the senior authors of the study, which appears in the Oct. 18 edition of the journal eLife. The other senior author is Weiping Han, head of the Laboratory of Metabolic Medicine at the Singapore Bioimaging Consortium in Singapore. Naiyan Chen, a postdoc at the Singapore Bioimaging Consortium and the McGovern Institute, is the lead author.

Turning on appetite

It has long been known that the hypothalamus, an almond-sized structure located deep within the brain, controls appetite as well as energy expenditure, body temperature, and circadian rhythms including sleep cycles. While performing studies on glial cells in other parts of the brain, Chen noticed that the hypothalamus also appeared to have a lot of glial cell activity.

“I was very curious at that point what glial cells would be doing in the hypothalamus, since glial cells have been shown in other brain areas to have an influence on regulation of neuronal function,” she says.

Within the hypothalamus, scientists have identified two key groups of neurons that regulate appetite, known as AgRP neurons and POMC neurons. AgRP neurons stimulate feeding, while POMC neurons suppress appetite.

Until recently it has been difficult to study the role of glial cells in controlling appetite or any other brain function, because scientists haven’t developed many techniques for silencing or stimulating these cells, as they have for neurons. Glial cells, which make up about half of the cells in the brain, have many supporting roles, including cushioning neurons and helping them form connections with one another.

In this study, the research team used a new technique developed at the University of North Carolina to study a type of glial cell known as an astrocyte. Using this strategy, researchers can engineer specific cells to produce a surface receptor that binds to a chemical compound known as CNO, a derivative of clozapine. Then, when CNO is given, it activates the glial cells.

The MIT team found that turning on astrocyte activity with just a single dose of CNO had a significant effect on feeding behavior.

“When we gave the compound that specifically activated the receptors, we saw a robust increase in feeding,” Chen says. “Mice are not known to eat very much in the daytime, but when we gave drugs to these animals that express a particular receptor, they were eating a lot.”

The researchers also found that in the short term (three days), the mice did not gain extra weight, even though they were eating more.

“This raises the possibility that glial cells may also be modulating neurons that control energy expenditures, to compensate for the increased food intake,” Chen says. “They might have multiple neuronal partners and modulate multiple energy homeostasis functions all at the same time.”

When the researchers silenced activity in the astrocytes, they found that the mice ate less than normal.

Suzanne Dickson, a professor of neuroendocrinology at the University of Gothenburg in Sweden described the study as part of a “paradigm shift” toward the idea that glial cells have a less passive role than previously believed.

“We tend to think of glial cells as providing a support network for neuronal processes and that their activation is also important in certain forms of brain trauma or inflammation,” says Dickson, who was not involved in the research. “This study adds to the emerging evidence base that glial cells may also exert specific effects to control nerve cell function in normal physiology.”

Unknown interactions

Still unknown is how the astrocytes exert their effects on neurons. Some recent studies have suggested that glial cells can secrete chemical messengers such as glutamate and ATP; if so, these “gliotransmitters” could influence neuron activity.

Another hypothesis is that instead of secreting chemicals, astrocytes exert their effects by controlling the uptake of neurotransmitters from the space surrounding neurons, thereby affecting neuron activity indirectly.

Feng now plans to develop new research tools that could help scientists learn more about astrocyte-neuron interactions and how astrocytes contribute to modulation of appetite and feeding. He also hopes to learn more about whether there are different types of astrocytes that may contribute differently to feeding behavior, especially abnormal behavior.

“We really know very little about how astrocytes contribute to the modulation of appetite, eating, and metabolism,” he says. “In the future, dissecting out these functional difference will be critical for our understanding of these disorders.”

Reading the rules of gene regulation

Anne Trafton |

We have a reasonable understanding of the rules behind the genome’s protein-coding components. We can look at a DNA sequence and point with confidence to where a gene’s coding region begins, where it ends, and pieces of its geography.

For the remaining 98 percent of the genome — the part that dictates which genes a cell reads — it’s a different story. What knowledge we have of the rules governing this “dark matter” comes from from studying and manipulating individual bits of noncoding DNA one at a time. The rulebook that governs how the noncoding genome works, however, has remained out of reach.

“Ninety percent of the genetic variations that affect human disease are in the noncoding regions,” said Broad founding director Eric Lander. “But we haven’t had any way to tell, in a systematic way, which regulators affect which genes.”

In a pair of newly published Science papers, two research teams at the Broad show how methods leveraging CRISPR gene editing could help grasp those rules.

Using two complementary approaches, the teams — one from the Lander lab, the other from that of Broad Core Institute Member and McGovern Institute for Brain Research investigator Feng Zhang — used CRISPR as a tool to systematically probe thousands of noncoding DNA sequences simultaneously (much as Zhang and others did previously with coding DNA). In the process, both identified several interesting genetic regulators, including ones millions of bases away from the genes they control.

“We’d like to be able to catalog the noncoding elements that control every gene’s expression in every cell type,” said Jesse Engreitz, a postdoctoral fellow in the Lander lab and senior author on one of the papers. “This is a massive problem in biology, and it’s a rate-limiting step for connecting many genetic associations to their fundamental molecular mechanisms and to human disease.”
Variations on a theme

Both teams used pooled CRISPR screens (which scan and edit large swaths of the genome simultaneously using a molecular scalpel called the Cas9 enzyme and thousands of guide RNAs, which target Cas9 to specific sequences) to perturb noncoding DNA. But they did so in different ways.

Zhang, Neville Sanjana (a Zhang lab alum and now a core member of the New York Genome Center), and Jason Wright (another Zhang alum, now at Homology Medicines) used Cas9 to make precise edits to overlapping stretches of noncoding DNA — in their case, in regions surrounding three genes (NF1, NF2, and CUL3) whose functional loss has been linked to drug resistance in a form of melanoma.

“This approach lets us induce a wide diversity of mutations,” Sanjana explained. “We don’t have to speculate how a given sequence might best be disrupted.”

Engreitz, Lander, and graduate student Charles Fulco, on the other hand, employed a CRISPR interference system, using an inactive or “dead” form of Cas9 fused to a protein fragment called a KRAB domain to silence their target sequences (around MYC and GATA1, the genes for two important transcription factors).

“This system provides a good quantitative estimate of a given noncoding region’s regulatory influence,” Engreitz said. “It both shows you where the dials are that control a given gene, and tells you how much each dial matters.”

Each team then used a functional readout (increased drug resistance in melanoma cells for Sanjana, Wright, and Zhang; a drop in cell growth for Fulco, Lander, and Engreitz) and deep sequencing to see which of their guide RNAs impacted expression of their genes of interest and map the regulators those guide RNAs affected.

The two teams’ findings, confirmed with an array of additional techniques (e.g., chromatin profiling, 3D conformational capture, transcription factor profiling), point to the potential for tracing the noncoding genome’s regulatory wiring leveraging CRISPR tools. Fulco, Lander, and Engreitz found and ranked the relative importance of seven MYC and three GATA1 enhancers (short pieces of noncoding DNA that boost a gene’s chances of being read). Sanjana, Wright, and Zhang’s screen pinpointed numerous enhancers and transcription factor binding sites just for CUL3 alone.
Studying sequences in their natural habitat

While similar in principle to traditional reporter assays (where scientists couple interesting sequences to reporter genes in plasmids), these pooled CRISPR screens have a distinct difference: they probe the sequences directly, in their native habitat.

“The screens interrogate the sequences in their endogenous context,” Sanjana emphasized. “Reporter assays can be very helpful, but they lack the 3D conformation or local chromatin environment of the native genomic context. Here, the regulatory sequences undergo all of their normal interactions.”

“For example, we could see long-range loops between gene promoters and noncoding sites thousands of bases away,” he continued. “We would have missed these interesting 3D interactions entirely if we just looked at these regulatory elements in isolation.”

One limitation, Engreitz noted, is that neither CRISPR approach, in its current form, addresses the genome’s inherent redundancy. “Maybe it’s not enough to break one enhancer to really understand how a gene is controlled. Maybe you have to break more than one,” he said. “We can’t do that yet.”

But Engreitz, Sanjana, and Lander are all optimistic about the potential for using CRISPR-based approaches to reveal the noncoding genome’s underlying order.

“One interesting challenge with the noncoding genome is that while it is huge, the individual functional elements within it can be quite small,” Sanjana said. “In the future, it will be important to think about how we can develop new approaches that interrogate larger regions while maintaining high resolution.”

Engreitz agreed, adding, “There’s a potential that as we map more of these connections we’re going to learn the rules that let us predict them for the rest of the noncoding genome.”

“These approaches, using libraries of guide RNAs to bring CRISPR in to cut or bring in inhibitors, let you directly see the effects of large areas of noncoding DNA on different genes,” Lander said. “I think this is going to crack open systematic maps of gene regulation.”

Papers cited:

Fulco CP et al. Systematic mapping of functional enhancer-promoter connections with CRISPR interference. Science. September 29, 2016. DOI: 10.1126/science.aag2445

Sanjana NE et al. High-resolution interrogation of functional elements in the noncoding genome. Science. September 29, 2016. DOI: 10.1126/science.aaf7613

Pinpointing a brain circuit that can keep fears at bay

Anne Trafton |

People who are too frightened of flying to board an airplane, or too scared of spiders to venture into the basement, can seek a kind of treatment called exposure therapy. In a safe environment, they repeatedly face cues such as photos of planes or black widows, as a way to stamp out their fearful response — a process known as extinction.

Unfortunately, the effects of exposure therapy are not permanent, and many people experience a relapse. MIT scientists have now identified a way to enhance the long-term benefit of extinction in rats, offering a way to improve the therapy in people suffering from phobias and more complicated conditions such as post-traumatic stress disorder (PTSD).

Work conducted in the laboratory of Ki Goosens, a research affiliate of the McGovern Institute for Brain Research, has pinpointed a neural circuit that becomes active during exposure therapy in the rats. In a study published Sept. 27 in eLife, the researchers showed that they could stretch the therapy’s benefits for at least two months by boosting the circuit’s activity during treatment.

“When you give extinction training to humans or rats, and you wait long enough, you observe a phenomenon called spontaneous recovery, in which the fear that was originally learned comes back,” Goosens explains. “It’s one of the barriers to this type of therapy. You spend all this time going through it, but then it’s not a permanent fix for your problem.”

According to statistics from the National Institute of Mental Health, 18 percent of U.S. adults are diagnosed with a fear or anxiety disorder each year, with 22 percent of those patients experiencing severe symptoms.

How to quench a fear

The neural circuit identified by the scientists connects a part of the brain involved in fear memory, called the basolateral amygdala (BLA), with another region called the nucleus accumbens (NAc), that helps the brain process rewarding events. Goosens and her colleagues call it the BLA-NAc circuit.

Researchers have been considering a link between fear and reward for some time, Goosens says. “The amygdala is a part of the brain that is tightly linked with fear memory but it’s also been linked to positive reward learning as well, and the accumbens is a key reward area in the brain,” she explains. “What we’ve been thinking about is whether extinction is rewarding. When you’re expecting something bad and you don’t get it, does your brain treat that like it’s a good thing?”

To find out if there was a specific brain circuit involved, the researchers first trained rats to fear a certain noise by pairing it with foot shock. They later gave the rats extinction training, during which the noise was presented in the absence of foot shock, and they looked at markers of neural activity in the brain. The results revealed the BLA-NAc reward circuit was recruited by the brain during exposure therapy, as the rats gave up their fear of the bad noise.

Once Goosens and her colleagues had identified the circuit, they looked for ways to boost its activity. First, they paired a sugary drink with the fear-related sound during extinction training, hoping to associate the sound with a reward. This type of training, called counterconditioning, associates fear-eliciting cues with rewarding events or memories, instead of with neutral events as in most extinction training.

Rats that received the counterconditioning were significantly less likely to spontaneously revert to their fearful states, compared to those that received regular extinction training for up to 55 days later, the scientists found.

They also found that the benefits of extinction could be prolonged with optogenetic stimulation, in which the circuit was genetically modified so that it could be stimulated directly with tiny bursts of light from an optical fiber.

The ongoing benefit that came from stimulating the circuit was one of the most surprising — and welcome — findings from the study, Goosens says. “The effect that we saw was one that really emerged months later, and we want to know what’s happening over those two months. What is the circuit doing to suppress the recovery of fear over that period of time? We still don’t understand what that is.”

Another interesting finding from the study was that the circuit was active during both fear learning and fear extinction, says lead author Susana Correia, a former research scientist in the Goosens lab who is now a scientist at Ironwood Pharmaceuticals. “Understanding if these are molecularly different subcircuits within this projection could allow the development of a pharmaceutical approach to target the fear extinction pathway and to improve cognitive therapy,” Correia says.

Immediate and future impacts on therapy

Some therapists are already using counterconditioning in treating PTSD, and Goosens suggests that the rat study might encourage further exploration of this technique in human therapy.

And while it isn’t likely that humans will receive direct optogenetic therapy any time soon, Goosens says there is a benefit to knowing exactly which circuits are involved in extinction.

In neurofeedback studies, for instance, brain scan technologies such as fMRI or EEG could be used to help a patient learn to activate specific parts of their brain, including the BLA-NAc reward circuit, during exposure therapy.

Studies like this one, Goosens says, offer a “target for a personalized medicine approach where feedback is used during therapy to enhance the effectiveness of that therapy.”

Other MIT authors on the paper include technical assistant Anna McGrath, undergraduate Allison Lee, and McGovern principal investigator and Institute Professor Ann Graybiel.

The study was funded by the U.S. Army Research Office, the Defense Advanced Research Projects Agency (DARPA), and the National Institute of Mental Health.