Category: News Briefs

In immune cells, X marks the spot(s)

By researching the effects of sex chromosomes on two types of immune cells, researchers in the Page Lab explore the biological underpinnings of sex biases in immunity and autoimmune disease

Greta Friar | Whitehead Institute
August 6, 2024

There are many known sex differences in health and disease: cases in which either men or women are more likely to get a disease, experience a symptom, or have a certain drug side effect. Some of these sex differences are caused by social and environmental factors: for example, when men smoked more than women, men were more likely to develop lung cancer. However, some have biological underpinnings. For example, men are more likely to be red-green colorblind because the relevant gene is on the X chromosome, of which men with XY chromosomes have no backup copy for a dysfunctional version.

Often, the specific factors contributing to a sex difference are hard to tease apart; there may not be a simple way to tell what is caused by sex chromosomes versus sex hormones versus environment. To address this question, researchers in Whitehead Institute Member David Page’s lab previously developed an approach to identify the contributions of the sex chromosomes to sex differences. Now, Page and former postdoc in his lab Laura Blanton have built on that work by measuring the effects of the sex chromosomes on two types of immune cells. The work, published in the journal Cell Genomics on August 6, shows that sex chromosome gene expression is consistent across cell types, but that its effects are cell type specific.

Sex differences are common in the function and dysfunction of our immune system. Examples include the typically weaker male immune response to pathogens and vaccines, and the female-biased frequency of autoimmune diseases. Page and Blanton’s work in immune cells examines several genes that have been implicated in such sex differences.

Developing a method to measure sex chromosome influence

The approach that the researchers used is based on several facts about sex chromosomes. Firstly, although females typically have two X chromosomes and males typically have one X and one Y, there are people with rare combinations of sex chromosomes, who have anywhere from 1-5 X chromosomes and 0-4 Y chromosomes. Secondly, there are two types of X chromosome: The active X chromosome (Xa) and the inactive X chromosome (Xi). They are genetically identical, but many of the genes on Xi are either switched off or have their expression level dialed way down.

Xa does not really function as a sex chromosome since everyone in the world has exactly one Xa regardless of their sex. In people with more than one X chromosome, any additional X chromosomes are always Xi. Furthermore, Page and Blanton’s research demonstrates that Xa responds to gene expression by Xi and Y—the sex chromosomes—in the same manner as do the other 22 pairs of non-sex chromosomes—the autosomes.

With these facts in mind, the researchers collected cells from donors with different combinations of sex chromosomes. Then they measured the expression of every gene in these cells, across the donor population, and observed how the expression of each gene changed with the addition of each Xi or Y chromosome.

This approach was first shared in a Cell Genomics paper by Page and former postdoc Adrianna San Roman in 2023. They had cultured two types of cells, fibroblasts and lymphoblastoid cell lines, from donor tissue samples. They found that the effects of Xi and Y were modular—each additional chromosome changed gene expression by about the same amount. This approach allowed the researchers to identify which genes are sensitive to regulation by the sex chromosomes, and to measure the strength of the effect for each responsive gene.

In that and a following paper, Page and San Roman looked at how Xi and Y affect gene expression from Xa and the autosomes. Blanton expanded the study of Xi and Y by using the same approach in two types of immune cells, monocytes and CD4+ T cells, taken directly from donors’ blood. Studying cells taken directly from the body, rather than cells cultured in the lab, enabled the researchers to confirm that their observations applied in both conditions.

In all three papers, the researchers found that the sex chromosomes have significant effects on the expression levels of many genes that are active throughout the body. They also identified a particular pair of genes as driving much of this effect in all four cell types. The genes, ZFX and ZFY, found on the X and Y chromosomes respectively, are transcription factors that can dial up the expression of other genes. The pair originates from the same ancestral gene, and although they have grown slightly apart since the X and Y chromosomes diverged, they still perform the same gene regulatory function. The researchers found that they tended to affect expression of the same gene targets by similar though not identical amounts.

In other words, the presence of either sex chromosome causes roughly the same effect on expression of autosomal and Xa genes. This similarity makes sense: carefully calibrated gene regulation is necessary in every body, and so each sex chromosome must maintain that function. It does, however, make it harder to spot the cases in which sex chromosomes contribute to sex differences in health and disease.

“Sex differences in health and disease could stem from the rare instances in which one gene responds very differently to Xi versus Y—we found cases where that occurs,” Blanton says. “They could also stem from subtle differences in the gene expression changes caused by Xi and Y that build up into larger effects downstream.”

Blanton then combined her and San Roman’s data in order to look at how the effects of sex chromosome dosage—how many Xs or Ys are in a cell—compared across all four cell types.

The effects of sex chromosomes on immune cells

 Blanton found that gene expression from the sex chromosomes was consistent across all four cell types. The exceptions to this rule were always X chromosome genes that are only expressed on Xa, and so could be regulated by Xi and Y in the way that autosomal genes are. This contrasts with speculation that different genes on Xi might be silenced in different cells.

However, each cell type had a distinct response to this identical sex chromosome gene expression. Different biological pathways were affected, or the same biological pathway could be affected in the opposite direction. Key immune cell processes affected by sex chromosome dosage in either monocytes or T cells included production of immune system proteins, signaling, and inflammatory response.

The cell type specific responses were due to different genes responding to the sex chromosomes in each cell type. The researchers do not yet know the mechanism causing the same gene to respond to sex chromosome dosage in one cell type but not another. One possibility is that access to the genes is blocked in some of the cell types. Regions of DNA can become tightly packed so that a gene, or a DNA region that regulates the gene, becomes inaccessible to transcription factors such as ZFX and ZFY, and so they cannot affect the gene’s expression. Another possibility is that the genes might require specific partner molecules in order for their expression level to increase, and that these partners may be present in one cell type but not the other.

Blanton also measured how X chromosome dosage affected T cells in their inactive state, when there is no perceived immune threat, versus their activated state, when they begin to produce an immune response and replicate themselves. Increases in X chromosome dosage led to heightened activation, with increased expression of genes related to proliferation. This finding highlights the importance of looking at how sex chromosomes affect not just different cell types, but cells in different states or scenarios.

“As we learn what pathways the sex chromosomes influence in each cell type, we can begin to make sense of the contributions of the sex chromosomes to each cell type’s functions and its roles in disease,” Blanton says.

Although Page and Blanton found that the presence of an Xi or Y chromosome had very similar effects on most genes, the researchers did identify one interesting case in which response to X and Y differed. FCG2RB is a gene involved in immunity that has been implicated in and thought to contribute to the female bias in developing systemic lupus erythematosus (SLE). Blanton found that unlike most genes, FCGR2B is sensitive to X and not Y chromosome dosage. This strengthens the case that higher expression of FCGR2B could be driving the SLE female bias.

“FCGR2B provides a promising opportunity to study the contributions of the sex chromosomes to a sex bias in disease, and to learn more about the biology of a chronic disease that affects many people around the world,” Page says.

In other cases, the researchers found that genes which have been suspected to contribute to female bias in disease did not have a strong response to X chromosome dosage. For example, TLR7 is thought to contribute to female bias in developing autoimmunity, and CD40LG is thought to contribute to female bias in developing lupus. Neither of the genes showed increased expression as X chromosome dosage increased. This suggests that other mechanisms may be driving the sex bias in these cases.

Because of the limited pool of donors, the researchers were not able to identify every gene that responds to sex chromosome dosage, and future research may uncover more sex-chromosome-sensitive genes of interest. Meanwhile, the Page lab continues to investigate the sex chromosomes’ shared role as regulators of gene expression throughout the body.

“We’ve got to recalibrate our thinking from the view that X and Y are mainly involved in differentiating males and females, to understanding that they also have largely shared functions that are important throughout the body,” Page says. “At the same time, I think that uncovering the biology of Xi is going to be incredibly important for understanding women’s health and sex differences in health and disease.”

News Brief: Lamason Lab uncovers seven novel effectors in Rickettsia parkeri infection

The enemy within: new research reveals insights into the arsenal Rickettsia parkeri uses against its host

Lillian Eden | Department of Biology
July 29, 2024

Identifying secreted proteins is critical to understanding how obligately intracellular pathogens hijack host machinery during infection, but identifying them is akin to finding a needle in a haystack.

For then-graduate student Allen Sanderlin, PhD ’24, the first indication that a risky, unlikely project might work was cyan, tic tac-shaped structures seen through a microscope — proof that his bacterial pathogen of interest was labeling its own proteins.  

Sanderlin, a member of the Lamason Lab in the Department of Biology at MIT, studies Rickettsia parkeri, a less virulent relative of the bacterial pathogen that causes Rocky Mountain Spotted Fever, a sometimes severe tickborne illness. No vaccine exists and definitive tests to diagnose an infection by Rickettsia are limited.

Rickettsia species are tricky to work with because they are obligately intracellular pathogens whose entire life cycles occur exclusively inside cells. Many approaches that have advanced our understanding of other bacterial infections and how those pathogens interact with their host aren’t applicable to Rickettsia because they can’t be grown on a plate in a lab setting. 

In a paper recently published in Nature Communications, the Lamason Lab outlines an approach for labeling and isolating R. parkeri proteins released during infection. This research reveals seven previously unknown secreted factors, known as effectors, more than doubling the number of known effectors in R. parkeri. 

Better-studied bacteria are known to hijack the host’s machinery via dozens or hundreds of secreted effectors, whose roles include manipulating the host cell to make it more susceptible to infection. However, finding those effectors in the soup of all other materials within the host cell is akin to looking for a needle in a haystack, with an added twist that researchers aren’t even sure what those needles look like for Rickettsia.  

Approaches that worked to identify the six previously known secreted effectors are limited in their scope. For example, some were found by comparing pathogenic Rickettsia to nonpathogenic strains of the bacteria, or by searching for proteins with domains that overlap with effectors from better-studied bacteria. Predictive modeling, however, relies on proteins being evolutionarily conserved. 

“Time and time again, we keep finding that Rickettsia are just weird — or, at least, weird compared to our understanding of other bacteria,” says Sanderlin, the paper’s first author. “This labeling tool allows us to answer some really exciting questions about rickettsial biology that weren’t possible before.”

The cyan tic tacs

To selectively label R. parkeri proteins, Sanderlin used a method called cell-selective bioorthogonal non-canonical amino acid tagging. BONCAT was first described in research from the Tirrell Lab at Caltech. The Lamason Lab, however, is the first group to use the tool successfully in an obligate intracellular bacterial pathogen; the thrilling moment when Sanderlin saw cyan tic-tac shapes indicated successfully labeling only the pathogen, not the host. 

Sanderlin next used an approach called selective lysis, carefully breaking open the host cell while leaving the pathogen, filled with labeled proteins, intact. This allowed him to extract proteins that R. parkeri had released into its host because the only labeled proteins amid other host cell material were effectors the pathogen had secreted. 

Sanderlin had successfully isolated and identified seven needles in the haystack, effectors never before identified in Rickettsia biology. The novel secreted rickettsial factors are dubbed SrfA, SrfB, SrfC, SrfD, SrfE, SrfF, and SrfG. 

“Every grad student wants to be able to name something,” Sanderlin says. “The most exciting — but frustrating — thing was that these proteins don’t look like anything we’ve seen before.”

Special delivery

Theoretically, Sanderlin says, once the effectors are secreted, they work independently from the bacteria — a driver delivering a pizza does not need to check back in with the store at every merge or turn.

Since SrfA-G didn’t resemble other known effectors or host proteins the pathogen could be mimicking during infection, Sanderlin then tried to answer some basic questions about their behavior. Where the effectors localize, meaning where in the cell they go, could hint at their purpose and what further experiments could be used to investigate it. 

To determine where the effectors were going, Sanderlin added the effectors he’d found to uninfected cells by introducing DNA that caused human cell lines to express those proteins. The experiment succeeded: he discovered that different Srfs went to different places throughout the host cells.  

SrfF and SrfG are found throughout the cytoplasm, whereas SrfB localizes to the mitochondria. That was especially intriguing because its structure is not predicted to interact with or find its way to the mitochondria, and the organelle appears unchanged despite the presence of the effector. 

Further, SrfC and SrfD found their way to the endoplasmic reticulum. The ER would be especially useful for a pathogen to appropriate, given that it is a dynamic organelle present throughout the cell and has many essential roles, including synthesizing proteins and metabolizing lipids. 

Aside from where effectors localize, knowing what they may interact with is critical. Sanderlin showed that SrfD interacts with Sec61, a protein complex that delivers proteins across the ER membrane. In keeping with the theme of the novelty of Sanderlin’s findings, SrfD does not resemble any proteins known to interact with the ER or Sec61. 

With this tool, Sanderlin identified novel proteins whose binding partners and role during infection can now be studied further. 

“These results are exciting but tantalizing,” Sanderlin says. “What Rickettsia secrete — the effectors, what they are, and what they do is, by and large, still a black box.” 

There are very likely other effectors in the proverbial cellular haystack. Sanderlin found that SrfA-G are not found in every species of Rickettsia, and his experiments were solely conducted with Rickettsia at late stages of infection — earlier windows of time may make use of different effectors. This research was also carried out in human cell lines, so there may be an entirely separate repertoire of effectors in ticks, which are responsible for spreading the pathogen.

Expanding Tool Development

Becky Lamason, the senior author of the Nature Communications paper, noted that this tool is one of a few avenues the lab is exploring to investigate R. parkeri, including a paper in the Journal of Bacteriology on conditional genetic manipulation. Characterizing how the pathogen behaves with or without a particular effector is leaps and bounds ahead of where the field was just a few years ago when Sanderlin was Lamason’s first graduate student to join the lab.

“What I always hoped for in the lab is to push the technology, but also get to the biology. These are two of what will hopefully be a suite of ways to attack this problem of understanding how these bacteria rewire and manipulate the host cell,” Lamason says. “We’re excited, but we’ve only scratched the surface.”

Unusual Labmates: Meet tardigrades, the crafters of nature’s ultimate survival kit

Whitehead Institute Member Siniša Hrvatin is studying tardigrades to decode the mechanisms enabling their survival in extreme environmental conditions. Learn about the biology of these microscopic “water bears” and what makes them a particularly fascinating model organism.

Shafaq Zia | Whitehead Institute
July 23, 2024

Tardigrades, also affectionately known as “water bears” or “moss piglets”, are remarkable microscopic organisms that have captured the imagination of scientists and nature enthusiasts alike.

With adults measuring anywhere from 0.2 to 1.2 millimeters in length — as big as a grain of salt — tardigrades possess the astounding ability to survive harsh environmental conditions. These resilient creatures have been found in habitats ranging from the depths of oceans and hot radioactive springs to the frigid expanses of Antarctica. It is their unparalleled adaptability that makes them invaluable as a model organism for researchers like Whitehead Institute Member Siniša Hrvatin, who’s studying physiological adaptation in animals with a focus on states that can slow down tissue damage, disease progression, and even aging.

Follow along to learn what’s behind tardigrades’ nearly indestructible nature, how researchers at Whitehead Institute — and beyond — are studying them, and what insights this work can offer into long-term organ preservation, space exploration, and more.

Big discovery of a tiny creature

In 1773, German naturalist Johann August Ephraim Goeze was analyzing moss samples under a microscope when he stumbled upon an unusual creature. Captivated by its peculiar appearance, he continued his observations and documented the discovery of Kleiner Wasserbär, translating to “little water bear”, in his publication. This work also featured the first-ever drawing of a tardigrade.

Since then, researchers’ understanding of this remarkable organism has evolved alongside advancements in imaging technology. Today, tardigrades are recognized as bilaterally symmetrical invertebrates with two eyes and eight chubby legs adorned with hook-like claws. Often described as a mix between nematodes and insects, these extremophiles are able to withstand freezing, intense radiation, vacuum of outer space, desiccation, chemical treatments, and possibly more.

And the best part? Despite their otherworldly appearance and surprising capabilities, tardigrades share plenty of similarities with larger, more complex organisms, including possessing a primordial brain, muscles, and even a digestive system.

The biology of an extremophile

Researchers trace the evolutionary origins of tardigrades back to panarthropods, a group that includes now-extinct worm-like organisms called lobopodians. To date, over a thousand species of tardigrades have been identified, with terrestrial species inhabiting environments like moss, leaf litter, and lichen, grassland, and deserts while aquatic ones are found in both fresh and saltwater.

Little is known about tardigrades’ diet but researchers are particularly drawn to herbivorous ones that like to munch on single-celled algae and thrive in water. There’s good reason for it: algae are inexpensive to grow in the lab with just light and basic nutrients. But it’s not just their diet that makes tardigrades an attractive model organism — they also have a short generation time (11 to 14 days), with eggs hatching within a four-day span. In fact, some species are able to reproduce without sexual reproduction through a process called parthenogenesis, during which the female egg undergoes cell division without fertilization by a male gamete.

Although genomic resources for studying tardigrades are limited to only a few species, researchers from Keio University and University of Edinburgh have successfully sequenced the genome of a moss-residing tardigrade commonly used in research called Hypsibius exemplaris. Its genome is less than half the size of a Drosophila melanogaster genome, consisting of 105 million base pairs that serve as the building blocks of DNA.

In spite of their small genome — and only a few thousand cells in the body — tardigrades have a well-defined miniaturized body plan, consisting of a head and four segments, that holds valuable insights for researchers looking to decode their adaptation prowess.

Inside tardigrade research at Whitehead Institute

In 2022, as Hrvatin was setting up his lab at Whitehead Institute, a question lingered in his mind. “I was trying to find animals that can survive being frozen for long periods of time and then continue living,” he says. “But there are not that many that fit the bill.”

Then, an undergraduate student at Massachusetts Institute of Technology (MIT) expressed her enthusiasm for astrobiology — the study of life across the universe — and highlighted tardigrades as a favorite among space researchers. Hrvatin was intrigued.

Up until this point, his research had centered upon two states of dormancy, or reduced metabolic activity, in animals: hibernation and a shorter, less intense torpor. But tardigrades possessed a survival mechanism unlike any other. When faced with harsh conditions like dehydration, they would expel water, retract their head and legs, and curl up in a small, dry ball, entering a state of suspended animation called crytobiosis or tun formation.

For decades, researchers hypothesized that the tun state might be responsible for tardigrades’ unparalleled ability to withstand a myriad of environmental assaults, including extremely low temperature. However, recent work has revealed that these animals utilize a separate and unique adaptation, distinct from the tun state, to survive being frozen for extended periods. In fact, preliminary evidence from a preprint by a team of scientists at UC Berkeley and UC San Francisco illustrates unique patterns of how tardigrades survive freezing while hydrated in water.

This phenomenon is markedly different from hibernation and its cousin torpor. “Unlike animals lowering their body temperature, we’re talking about putting tardigrades at minus 180 degrees Celsius, and then thawing them,” says Hrvatin. In fact, cryobiosis is so intense that tardigrades’ metabolic activity drops to undetectable levels, rendering them virtually, but not quite, dead. The organisms can then remain in this state from months to years, only to revive as healthy when conditions become favorable once again.

Frozen in time

In 2014, a group of Japanese researchers at Tokyo’s National Institute for Polar Research undertook an intriguing experiment. They began by thawing moss samples collected from East Antarctica in November 1983. Then, they carefully teased apart each sample using tweezers to retrieve tardigrades that might be nestled within. Among the tardigrades the researchers found, two stood out: Sleeping Beauty 1 and Sleeping Beauty 2 who were believed to be undergoing cold induced-dormancy. Turns out, the researchers were right — within the first day of being placed in the Petri dish with water, the tardigrades began exhibiting slow movements despite having been frozen for over 30 years.

The Swiss army knife in tardigrades’ toolbox

Yet, the remarkable resilience of tardigrades continues to baffle scientists. Recently, they’ve uncovered what could be another potential weapon in the creatures’ arsenal: intrinsically disordered proteins or IDPs. Picture them as putty — a group of proteins that do not have a well-defined three-dimensional structure and can interact with other molecules to produce a range of different outcomes. Some researchers have linked these tardigrade-specific IDPs to the animals extraordinary resilience: under extreme heat, these proteins remain stable. And when desiccated, they form protective glasses that shield cells and vital enzymes from dehydration.

If confirmed, the implications of this work would extend beyond tardigrades’ survival, potentially revolutionizing dry vaccine storage and the development of drought-resistant crops.

Gene silencing tool has a need for speed

Small changes in the molecular machines that carry out RNA interference can lead to big differences in the efficacy of gene silencing. These new findings from the Bartel Lab have implications for the design of gene-silencing therapeutics.

Greta Friar | Whitehead Institute
July 17, 2024

RNA interference (RNAi) is a process that many organisms, including humans, use to decrease the activity of target RNAs in cells by triggering their degradation or slicing them in half. If the target is a messenger RNA, the intermediary between gene and protein, then RNAi can decrease or completely silence expression of the gene. Researchers figured out how to tailor RNAi to target different RNAs, and since then it has been used as a research tool to silence genes of interest. RNAi is also used in a growing number of therapeutics to silence genes that contribute to disease.

However, researchers still do not understand some of the biochemistry underlying RNAi. Slight differences in the design of the RNAi machinery can lead to big differences in how effective it is at decreasing gene expression. Through trial and error, researchers have worked out guidelines for making the most effective RNAi tools without understanding exactly why they work. However, Whitehead Institute Member David Bartel and graduate student in his lab Peter Wang have now dug deeper to figure out the mechanics of the main cellular machine involved in RNAi. The researchers’ findings, shared in Molecular Cell on July 17, not only provide explanations for some of the known rules for RNAi tool design, but also provide new insights that could improve future designs.

Slicing speed is highly variable

The cellular machine that carries out RNAi has two main parts. One is a guide RNA, a tiny RNA typically only 22 bases or nucleotides long. RNA, like DNA, is made of four possible bases, although RNA has the base uracil (U) instead of the DNA base thymine (T). RNA bases bind to each other in certain pairings—guanines (G) pair to cytosines (C) and adenines (A) pair to U’s—and the sequence of bases in the guide RNA corresponds to a complementary sequence within the target RNA. When the guide RNA comes across a target, the corresponding bases pair up, binding the RNAs. Then the other part of the RNAi machine, an Argonaute protein bound to the guide RNA, can slice the target RNA in half or trigger the cell to break it down more gradually.

In humans, AGO2 is the Argonaute protein that is best at slicing. Only a couple dozen RNA targets actually get sliced, but these few targets play essential roles in processes such as neuron signal control and accurate body shape formation. Slicing is also important for RNAi tools and therapeutics.

In order for AGO2 to slice its target, the target must be in the exact right position. As the guide and target RNAs bind together, they go through a series of motions to ultimately form a double helix. Only in that configuration can AGO2 slice the target.

Researchers had assumed that AGO2 slices through different target RNAs at roughly the same rate, because most research into this process used the same few guide RNAs. These guide RNAs happen to have similar features, and so similar slicing kinetics—but they turn out not to be representative of most guide RNAs.

Wang paired AGO2 with a larger variety of guide RNAs and measured the rate at which each AGO2-guide RNA complex sliced its targets. He found big differences. Whereas the commonly used guide RNAs might differ in their slicing rate by 2-fold, the larger pool of guide RNAs differed by as much as 250-fold. The slicing rates were often much slower than the researchers expected. Previously, researchers thought that all targets could be sliced relatively quickly, so the rate wasn’t considered as a limiting factor – other parts of the process were thought to determine the overall pace – but Wang found that slicing can sometimes be the slowest step.

“The important consideration is whether the slicing rate is faster or slower than other processes in the cell,” Wang says. “We found that for many guide RNAs, the slicing rate was the limiting factor. As such, it impacted the efficacy.”

The slower AGO2 is to slice targets, the more messenger RNAs will remain intact to be made into protein, meaning that the corresponding gene will continue being expressed. The researchers observed this in action: the guide RNAs with slower slicing rates decreased target gene expression by less than the faster ones.

Small changes lead to big differences in slicing rate

Next, the researchers explored what could be causing such big differences in slicing rate between guide RNAs. They mutated guide RNAs to swap out single bases along the guide RNA’s sequence—say, switching the 10th base in the sequence from a C to an A—and measured how this changed the slicing rate.

“The important consideration is whether the slicing rate is faster or slower than other processes in the cell,” Wang says. “We found that for many guide RNAs, the slicing rate was the limiting factor. As such, it impacted the efficacy.”

The researchers found that slicing rate increased when the base at position 7 was an A or a U. The bases A and U pair more weakly than C and G. The researchers found that having a weak A-U pair at that position, or a fully mismatched pair at position 6 or 7, may allow a kink to form in the double helix shape that actually makes the target easier to slice. Wang also found that slicing rate increases with certain substitutions at the 10th and the 17th base positions, although the researchers could not yet determine possible underlying mechanisms.

These observations correspond to existing recommendations for RNAi design, such as not using a G at position 7. The new work demonstrates that the reason these recommendations work is because they affect the slicing rate, and, in the case of position 7, the new work further identifies the specific mechanism at play.

Interplay between regions matters

People designing synthetic guide RNAs thought that the bases at the tail end, past the 16th position, were not very important. This is because in the case of the most commonly used guide RNAs, the target will be rapidly cleaved even if all of the tail end positions are mismatches that cannot pair.

However, Wang and Bartel found that the identity of the tail end bases are only irrelevant in a specific scenario that happens to be true of the most commonly used guide RNAs: when the bases in the center of the guide RNA (positions 9-12) are strong-pairing Cs and Gs. When the center pairings are weak, then the tail end bases need to be perfect matches to the target RNA. The researchers found that guide RNAs could have up to a 600-fold difference in tolerance for tail end mismatches based on the strength of their central pairings.

The reason for this difference has to do with the final set of motions that the two RNAs must perform in order to assume their final double helix shape. A perfectly paired tail end makes it easier for the RNAs to complete these motions. However, a strong enough center can pull the RNAs into the double helix even if the tail ends are not ideally suited for doing so.

The observation that weak central pairing requires perfect or near perfect tail end matches could provide a useful new guideline for designing synthetic RNAs. Any guide RNA runs the risk of sometimes binding other messenger RNAs that are similar enough to the intended target RNA. In the case of a therapy, this off-target binding can lead to negative side effects. Bartel and Wang suggest that researchers could design guide RNAs with weak centers, which would require more perfect pairing in the tail end, so that the guide RNA will be less likely to bind non-target RNAs; only the perfect pairing of the target’s RNA sequence would suffice.

Altogether, Wang and Bartel’s findings explain how small differences between guide RNAs can make such large differences in the efficacy of RNAi, providing a rationale for the long-standing RNAi design guidelines. Some of the findings even suggest new guidelines that could help with future synthetic guide RNA designs.

“Discovering the interplay between the center and tail end of the guide RNA was unexpected and satisfying,” says Bartel, who is also a professor at the Massachusetts Institute of Technology and a Howard Hughes Medical Investigator. “It explains why, even though the guidelines suggested that tail-end sequence doesn’t matter, the target RNAs that are sliced in our cells do have pairing to the tail end. This observation could prove useful to reduce off-target effects in RNAi therapeutics.”

A genome-wide screen in live hosts reveals new secrets of parasite infection

Researchers in the Lourido Lab performed the first genome-wide screen of Toxoplasma gondii in live hosts, revealing genes that are important for infection but previously undetected in cell culture experiments. 

Greta Friar | Whitehead Institute
July 8, 2024

Apicomplexan parasites are a common cause of disease, infecting hundreds of millions of people each year. They are responsible for spreading malaria; cryptosporidiosis – a severe childhood diarrheal disease; and toxoplasmosis – a disease that endangers immune compromised people and fetuses, and is the reason why pregnant women are told to avoid changing cat litter. Apicomplexan parasites are very good at infecting humans and many other animals, and persisting inside of them. The more that researchers can learn about how apicomplexans infect hosts, the better they will be able to develop effective treatments against the parasites.

To this end, researchers in Whitehead Institute Member Sebastian Lourido’s lab, led by graduate student Christopher Giuliano, have now completed a genome-wide screen of the apicomplexan parasite Toxoplasma gondii (T. gondii), which causes toxoplasmosis, during its infection of mice. This screen shows how important each gene is for the parasite’s ability to infect a host, providing clues to genes’ functions. In the journal Nature Microbiology on July 8, the researchers share their approach for tracing lineages of parasites in a live host, and some specific findings of interest—including a possible anti-parasitic drug target.

From dish to animal

Researchers in Lourido’s lab previously developed a screen to test the function of every T. gondii gene in cells in a dish in 2016. They used CRISPR gene editing technology to make mutant parasites in which each lineage had one gene inactivated. The researchers could then assess the importance of each gene to a parasite’s fitness, or ability to thrive, based on how well the mutants missing that gene did. If a mutant died off, this implied that its inactivated gene is essential for the parasite’s survival.

This screen taught the researchers a lot about T. gondii’s biology but faced a common limitation: the parasites were studied in a dish rather than a live host. Cell culture provides an easier way to study parasites, but the conditions are not the same as what parasites face in an animal host. A host’s body is a more complex and dynamic environment, so it may require parasites to rely on genes that they don’t need in the artificial setting of cell culture.

To overcome this limitation, researchers in Lourido’s lab figured out how to repeat the T. gondii genome-wide screen, which their colleagues in the lab had previously done in cell culture, in live mice. This was a massive undertaking, which required solving various technical challenges and running a large number of parallel experiments. T. gondii has around eight thousand genes, so the researchers performed pooled experiments, with each mouse getting infected by many different mutants—but not so many as to overwhelm the mouse. This meant that the researchers needed a way to more closely monitor the trajectories of mutants in the mouse. They needed to track the lineages of parasites that carried the same mutation over time, as this would allow them to see how different replicate lineages of a particular mutant performed.

“This is an outstanding resource,” says Lourido. “The results of the screen reveal such a broader spectrum of ways in which the parasites are interacting with hosts, and enrich our perception of the parasites’ abilities and vulnerabilities.”
The researchers added barcodes to the CRISPR tools that inactivated a gene of interest in the parasite. When they harvested the parasites’ descendants, the barcode would identify the lineage, distinguishing replicate parasites that had been mutated in the same way. This allowed the researchers to use a population-based analytical approach to rule out false results and decrease experimental noise. Then they could draw conclusions about how well each lineage did. Lineage tracing allowed them to map how different populations of parasites traveled throughout the host’s body, and whether some populations grew better in one organ versus another.

The researchers found 237 genes that contribute to the parasite’s fitness more in a live host than in cell culture. Many of these were not previously known to be important for the parasite’s fitness. The genes identified in the current screen are active in different parts of the parasite, and affect diverse aspects of its interactions with a host. The researchers also found instances in which parasite fitness in a live host increased when a gene was inactivated; these genes may be, for example, related to signals that the host immune system uses to detect the parasites. Next, the researchers followed up on several of the fitness-improving genes that stuck out as of particular interest.

Genes that make the difference in a live host

One gene that stuck out was GTP cyclohydrolase I (GCH), which codes for an enzyme involved in the production of the essential nutrient folate. Apicomplexans rely on folate, and so the researchers wanted to understand GCH’s role in securing it for the parasite. Cell culture media contains high levels of folate, and in this nutrient-rich environment, GCH is not essential. However, in a live mouse, the parasite must both scavenge folate and synthesize it using the metabolic pathway containing GCH. Lourido and Giuliano uncovered new details of how that pathway works.

Although previously GCH’s role was not fully understood, the importance of folate for apicomplexans is a well-known vulnerability that has been used to design anti-parasitic therapies. The anti-folate drug pyrimethamine was commonly used to treat malaria, but many parasites have developed resistance to it.

Some drug-resistant apicomplexans have increased the number of GCH gene copies that they have, suggesting that they may be using GCH-mediated folate synthesis to overcome pyrimethamine. The researchers found that combining a GCH inhibitor with pyrimethamine increased the efficacy of the drug against the parasites. The GCH inhibitor was also effective on its own. Unfortunately, the currently available GCH inhibitor targets mammalian as well as parasitic folate pathways, and so is not safe for use in animals. Giuliano and colleagues are working on developing a GCH inhibitor that is parasite-specific as a possible therapy.

“There was an entire half of the folate metabolism pathway that previously looked like it wasn’t important for parasites, simply because people add so much folate to cell culture media,” Giuliano says. “This is a good example of what can be missed in cell culture experiments, and what’s particularly exciting is that the finding has led us to a new drug candidate.”

Another gene of interest was RASP1. The researchers determined that RASP1 is not involved in initial infection attempts, but is needed if the parasites fail and need to mount a second attempt. They found that RASP1 is needed to reload an organelle of the parasites called a rhoptry that the parasites use to breach and reprogram host cells. Without RASP1, the parasites could only deploy one set of rhoptries, and so could only attempt one invasion.

Identifying the function of RASP1 in infection also demonstrated the importance of studying how parasites interact with different cell types. In cell culture, researchers typically culture parasites in fibroblasts, a connective tissue cell. The researchers found that parasites could invade fibroblasts with or without RASP1, suggesting that this cell type is easy for them to invade. However, when the parasites tried to invade macrophages, an immune cell, those without RASP1 often failed, suggesting that macrophages present the parasites with more of a challenge, requiring multiple attempts. The screen uncovered other probable cell-type specific pathways, which would not have been found using only model cell types in a dish.

The screen also highlighted a previously unnamed gene that the researchers are calling GRA72. Previous studies suggested that this gene plays a role in the vacuole or protective envelope that the parasite forms around itself. The Lourido lab researchers confirmed this, and discovered additional details of how the absence of GRA72 disrupts the parasite vacuole.

A rich resource for the future

Lourido, Giuliano, and colleagues hope that their findings will provide new insights into parasite biology and, especially in the case of GCH, lead to new therapies. They intend to continue pulling from the treasure trove of results—their screen identified many other genes of interest that require follow-up—to learn more about apicomplexan parasites and their interactions with mammalian hosts. Lourido says that other researchers in his lab have already used the results of the screen to guide them towards relevant genes and pathways in their own projects.

“This is an outstanding resource,” says Lourido, who is also an associate professor of biology at MIT. “The results of the screen reveal such a broader spectrum of ways in which the parasites are interacting with hosts, and enrich our perception of the parasites’ abilities and vulnerabilities.”

“Vaults” within germ cells offer more than safekeeping

Ribonucleoprotein (RNP) granules are believed to preserve maternal mRNA within eggs and developing embryos. The Lehman Lab reveals that a specific type of RNP granule also plays an active role in translating the mRNA that is crucial for specifying germ cells.

Shafaq Zia | Whitehead Institute
July 2, 2024

Maternal messenger RNAs (mRNAs), located within the cytoplasm of an immature egg, are crucial for jump starting development. Following fertilization, these mRNAs are passed onto the zygote, the first newly formed cell. Having been read from the maternal DNA genetic code, they serve as the sole templates for protein production essential for early development until the zygote’s own genes become active and take over.

Many maternal mRNAs are stored in ribonucleoprotein (RNP) granules, which are a type of membrane-less compartments, or condensates, within eggs and developing embryos. These granules are believed to preserve the mRNA in a “paused” state until the encoded proteins are needed for specific developmental processes upon fertilization of the egg cell. Then, certain developmental signals kick in to instruct the RNP granules to release the stored mRNA so the instructions can be translated into a functional protein.

One type of RNP granules called germ granules is found in embryo germplasm, a cytoplasmic region that gives rise to germ cells, which become the eggs or sperms of adult flies. Whitehead Institute Director Ruth Lehmann studies how germ cells form and transmit their genetic information across generations. Her lab is particularly interested in understanding how germ granules in embryos localize and regulate maternal mRNAs.

Now, Lehmann, along with graduate student Ruoyu Chen and colleagues, has uncovered that the role of germ granules in fruit flies (Drosophila melanogaster) extends beyond safeguarding maternal mRNAs. Their findings, published in the journal Nature Cell Biology on July 4, demonstrate that germ granules also play an active role in translating, or making into protein, a specific maternal mRNA, called nanos, crucial for specifying germ cells and the abdomen of the organism.

“Traditionally, scientists have thought of RNP granules as a dead zone for translation,” says Chen. “But through high-resolution imaging, we’ve challenged this notion and shown that the surface of these granules is actually a platform for translation of nanos mRNA.”

RNP granules act as vaults

Within a developing embryo, various fate-determining proteins dictate whether a cell will become a muscle, nerve, or skin cell in a fully-formed body. Nanos, a gene with conserved function in Drosophila and humans, guides the production of Nanos protein which instructs cells to develop into germline. Mutations in the nanos gene cause sterility in animals.

During early embryonic development, Nanos protein also helps establish the body plan of the fruit fly embryo — it specifies the posterior end or abdominal region, and guides the ordered development of tissues along the length of the body, from head to tail. In embryos with impaired Nanos function, the consequences are fatal.

“When Nanos protein isn’t functioning properly, the fruit fly embryos are really short,” says Chen. “This is because the embryo has no abdomen, which is basically half of its body. Nanos also has a second function that is conserved from flies to humans. This function is very local and instructs the cells with lots of Nanos to become germ cells. ”

Given Nanos’ vital role, embryos must safeguard instructions for its production until the embryo reaches a specific stage of development, when it is time to define the posterior region. Previous work has indicated that germ granules in the germplasm and germ cells can act like vaults, shielding the nanos mRNA from degradation or premature translation.

However, while the mRNA instructions for building the protein are distributed throughout the embryo, Nanos protein is found only in regions where germ granules reside. The mRNA does not get translated elsewhere in the embryo because of a regulatory protein called Smaug, named after the golden dragon depicted in J. R. R. Tolkien’s 1937 novel The Hobbit. Smaug binds to a non-protein coding segment of the mRNA known as the 3’ untranslated region (3’ UTR), extending beyond the protein-coding sequence, effectively suppressing the translation process.

For Lehmann, Chen, and their colleagues, this hinted at an intriguing relationship between nanos mRNA and germ granules. Are the granules essential for translating nanos mRNA into a functional protein? And if they are, is their role primarily to serve as a safekeeping place to evade repression by Smaug or do they actively facilitate the translation of nanos mRNA too?

To answer these questions, the researchers combined high-resolution imaging with a technique called the SunTag system to directly visualize the translation of nanos mRNA within Drosophila germ granules at the single-molecule level.

Unlike green fluorescent protein tagging, where a single fluorescent molecule is used, the SunTag system allows scientists to recruit multiple GFP copies for an amplified signal. First, a small protein tag, known as the SunTag, is fused with the protein-producing region of the nanos mRNA. As the mRNA instructions undergo translation, GFP molecules stick to the newly synthesized SunTag-Nanos protein, resulting in a bright fluorescent signal. Overlying this translation signal with fluorescent probes specifically labeling the mRNA then allows researchers to precisely visualize and track when and where the translation process is taking place.

“Using this system, we’ve discovered that when nanos mRNA is translated, it protrudes slightly from the surface of the granules like snakes peeking out of a box,” says Chen. “But they can’t fully emerge; a part of their sequence, specifically their “back” end, the 3’ UTR, remains tucked inside the granules. When the RNA is not translated, like during oogenesis, the tip coils back and is hidden inside the granule.”

With their high-resolution SunTag imaging technique, Lehmann, Chen and their colleagues have directly added to the work of other researchers with similar observations: mRNAs in the process of translation are in an extended configuration, while the 5’UTR curls back to the 3’UTR when the mRNAs are repressed.

Flipping on nanos translation

Then, the researchers went on to take a closer look at how these granules help initiate translation, while Smaug is able to inhibit the same nanos mRNA molecules from being translated in other areas of the embryo. They hypothesized that the untranslated region (UTR) of nanos mRNA, which remains concealed within the granules, might be playing a pivotal role in the translation process by localizing the mRNA instructions within germ cell granules. This localization, they speculated, protects the mRNA from Smaug’s inhibitory actions and facilitates Nanos protein production, so the posterior region can develop properly.

However, counter-intuitive to a simple protection model, they found that rather than being depleted, Smaug is enriched within germ granules, indicating that additional mechanisms within the RNP granule must counteract Smaug’s inhibitory effects. To explore this, the researchers turned to another regulatory protein called Oskar, which is known to interact with Smaug.

Discovered by Lehmann in a 1986 study, and named after a character in the German novel The Tin Drum, the oskar gene in Drosophila is known to help with the development of the posterior region. Later research has revealed that, during the development of oocytes, Oskar acts as a scaffold protein by initiating the formation of germ granules in germ cells and directing mRNA molecules, including nanos, towards the granules.

To gain a deeper understanding of Oskar’s full role in translational regulation in germ granules and its interaction with Smaug, the researchers engineered a modified version of Oskar protein. This altered Oskar protein retained its ability to initiate the formation of germ granules and localize nanos mRNA within them. However, Smaug no longer localized to the germ granules assembled by this altered Oskar.

The researchers then studied whether the mutant protein had any effect on nanos mRNA translation. In the germ cells with this mutant version of Oskar, the researchers saw a significant reduction in the translation of nanos mRNA. These findings, combined, suggested that Oskar regulates nanos translation in fruit fly embryos by recruiting Smaug to the granules and then counteracting its repression of translation.

“Condensates composed of RNAs and proteins are found in the cytoplasm of pretty much every cell and are thought to mediate mRNA storage or transport,” says Lehmann, who is also a professor of biology at the Massachusetts Institute of Technology. “But our results provide new insights into condensate biology by suggesting that condensates can be also used to specifically translate stored mRNAs.”

Indeed, in the oocyte, the germ granules are silent and only become activated when the egg is fertilized.

“This suggests that there might also be other ‘on and off switches’ governing translation within condensates during early development,” adds Lehmann. “How this is achieved and whether we could engineer this to happen at will in these and other granules is a question for the future.”

She’s fighting to stop the brain disease that killed her mother before it gets her

Jonathan Weissman is the senior author on a recent study on silencing a prion protein's expression. Prions cause devastating neurodegenerative disorders such as dementia, Huntington's, Parkinson's, and Lou Gehrig's disease. Silencing genes represents a step towards a therapeutic model for treating these diseases in humans.

Karen Weintraub | USA TODAY
June 27, 2024

CAMBRIDGE, Mass. ‒ Sonia Vallabh watched helplessly as her 51-year-old mother rapidly descended into dementia and died. It didn’t take long for Vallabh to realize she was destined for the same rare genetic fate.

Vallabh and her husband did what anyone would want to do in their situation: They decided to fight.

Armed with little more than incredible intellect and determination they set out to conquer her destiny.

A dozen years later, they’ve taken a major step in that direction, finding a way to shut off enough genetic signals to hold off the disease.

And in the process of trying to rescue Vallabh, they may save many, many others as well.

In a paper published Thursday in the prestigious journal Science, Vallabh and her husband, Eric Minikel, and their co-authors offer a way to disrupt brain diseases like the one that killed her mother.

The same approach should also work against diseases such as Huntington’s, Parkinson’s, Lou Gehrig’s disease and even Alzheimer’s, which result from the accumulation of toxic proteins. If it works as well as they think, it could also be useful against a vast array of other diseases that can be treated by shutting off genes.

“It doesn’t have to be the brain. It could be the muscles. It could be the kidneys. It could be really anywhere in the body where we have not easily been able to do these things before,” said Dr. Kiran Musunuru, a cardiologist and geneticist at the University of Pennsylvania’s Perelman School of Medicine, who wasn’t involved in the research but wrote a perspective accompanying the paper.

So far, they’ve proven it only in mice.

“The data are good as far as they go,” Vallabh said this week from her office at the Broad Institute of Harvard and the Massachusetts Institute of Technology, where she has worked since getting a Ph.D. at Harvard. She had already gotten a law degree from the university, but she and Minikel, then a transportation planner, both pursued biology degrees after her mother’s death. Now, they work together at the Broad.

“We’re far from this being a drug,” Vallabh said. “There’s always, always reason for caution. Sadly, everything is always more likely to fail than succeed.

“But there is justifiable reason for optimism.”

A terrible disease

The disease that killed Vallabh’s mother was one of a group of conditions called prion diseases. These include mad cow disease, which affects mostly cattle, scrapie, which affects sheep, and Creutzfeldt-Jakob disease, which kills about 350 Americans a year ‒ most within months of their first symptom.

These diseases are triggered when the prion protein found in all normal brains starts misfolding for some reason, as yet unknown.

“Prion disease can strike anybody,” Vallabh said, noting the 1 in 6,000 risk to the general population.

Though prion diseases are, in some cases, contagious, a federal study earlier this year concluded that chronic wasting disease, found in deer, elk and moose, is very unlikely to pass to people who eat the meat of sick animals.

In Vallabh’s case, the cause is genetic. Vallabh discovered after her mother’s death that she carries the same variant of the same gene that caused her mother’s disease, meaning she will certainly develop it.

The only question is when.

“The age of onset is extremely unpredictable,” Vallabh said. “Your parent’s age of onset doesn’t actually predict anything.”

How the gene-editing tool works

Vallabh and Minikel approached colleagues at the Whitehead Institute a biomedical research institute next to the Broad. They asked to collaborate on a new gene-editing approach to turn off Vallabh’s disease gene. The technique developed by Whitehead scientists is called CHARM (for Coupled Histone tail Autoinhibition Release of Methyltransferase).

While previous gene-editing tools have been described as scissors or erasers, Musunuru described CHARM as volume control, allowing scientists to tune a gene up or down. It has three advantages over previous strategies, he said.

The device is tiny, so it fits easily inside the virus needed to deliver it. Other gene-editing tools, like CRISPR, are bigger, which means they need to be broken into pieces and much more of the virus is needed to deliver those pieces to the brain, risking a dangerous immune reaction.

CHARM, Musunuru said, is “easier to deliver to hard-to-deliver spaces like the brain.”

At least in the mouse, it also seems to have reached throughout the brain, making the desired genetic change without other, unwanted ones, Musunuru said.

And finally, the research team figured out a way to turn the gene editor off after its work was done. “If it’s sticking around, there’s the potential for genetic mischief,” Musunuru said.

One shot on goal

While researchers, including Vallabh, continue to work to perfect an approach, the clock for Vallabh and others is ticking.

Right now there’s no viable treatment and if it takes too long to develop one, Vallabh will miss her window. Once the disease process starts, like a runaway train, it’ll be much harder to stop than it would be to just shut the gene off in the first place.

The more prion protein in the brain, the more likely it is to misfold. And the more likely it is for the disease to spread, a process that co-opts the natural form of the protein and converts it to the toxic form.

That’s why getting rid of as much of it as possible makes sense, said Jonathan Weissman, the senior author of the study, who leads a Whitehead lab.

“The biology is really clear. The need (for a cure) is so compelling,” Weissman said.

Every cell in the brain has the gene for making the prion protein. By silencing even 50% of those genes, Weissman figures he can prevent the disease. In mice, CHARM silenced up to 80% to 90%.

“We’ve figured out what to deliver. Now we have to figure out how to deliver it,” he said.

Another of the paper’s co-authors, the Broad’s Ben Deverman, published a study late last year showing he could deliver a gene-therapy-carrying virus throughout the brain. Others are developing other viral delivery systems.

Vallabh and Minikel have hedged their bets, helping to develop a so-called antisense oligonucleotide, or ASO, which uses another path for stopping the gene from making the prion protein.

The ASO, which is in early trials in people by a company called Ionis Pharmaceuticals, requires regular treatment rather than the one-and-done of gene therapy. Recruitment for that trial had to be paused in April because the number of would-be volunteers outstripped the available slots.

Vallabh isn’t ready yet to start any treatment yet herself.

“She has one shot on goal,” Musunuru said. “At some point, she’ll have to decide what’s the best strategy.”

In the meantime, the clock Vallabh can’t see continues to tick toward the onset.

She and Minikel stay exceedingly busy with their research along with their daughter, almost 7, and 4-year-old son ‒ both born via IVF and preimplantation genetic testing to ensure they wouldn’t inherit her genetic curse. (They were super lucky, Vallabh notes, to be living in Massachusetts where IVF is at least “approachable” financially.)

“There is a mountain ahead of us,” Vallabh said of the path to a cure. “There’s still a lot of hurdles, there’s still a lot to figure out.”

CHARMed collaboration creates a potent therapy candidate for fatal prion diseases

A new gene-silencing tool shows promise as a future therapy against prion diseases and paves the way for new approaches to treating disease.

Greta Friar | Whitehead Institute
June 27, 2024

Drug development is typically slow: The pipeline from basic research discoveries that provide the basis for a new drug to clinical trials and then production of a widely available medicine can take decades. But decades can feel impossibly far off to someone who currently has a fatal disease. Broad Institute of MIT and Harvard Senior Group Leader Sonia Vallabh is acutely aware of that race against time, because the topic of her research is a neurodegenerative and ultimately fatal disease — fatal familial insomnia, a type of prion disease — that she will almost certainly develop as she ages.

Vallabh and her husband, Eric Minikel, switched careers and became researchers after they learned that Vallabh carries a disease-causing version of the prion protein gene and that there is no effective therapy for fatal prion diseases. The two now run a lab at the Broad Institute, where they are working to develop drugs that can prevent and treat these diseases, and their deadline for success is not based on grant cycles or academic expectations but on the ticking time bomb in Vallabh’s genetic code.

That is why Vallabh was excited to discover, when she entered into a collaboration with Whitehead Institute for Biomedical Research member Jonathan Weissman, that Weissman’s group likes to work at full throttle. In less than two years, Weissman, Vallabh, and their collaborators have developed a set of molecular tools called CHARMs that can turn off disease-causing genes such as the prion protein gene — as well as, potentially, genes coding for many other proteins implicated in neurodegenerative and other diseases — and they are refining those tools to be good candidates for use in human patients. Although the tools still have many hurdles to pass before the researchers will know if they work as therapeutics, the team is encouraged by the speed with which they have developed the technology thus far.

“The spirit of the collaboration since the beginning has been that there was no waiting on formality,” Vallabh says. “As soon as we realized our mutual excitement to do this, everything was off to the races.”

Co-corresponding authors Weissman and Vallabh and co-first authors Edwin Neumann, a graduate student in Weissman’s lab, and Tessa Bertozzi, a postdoc in Weissman’s lab, describe CHARM — which stands for Coupled Histone tail for Autoinhibition Release of Methyltransferase — in a paper published today in the journal Science.

“With the Whitehead and Broad Institutes right next door to each other, I don’t think there’s any better place than this for a group of motivated people to move quickly and flexibly in the pursuit of academic science and medical technology,” says Weissman, who is also a professor of biology at MIT and a Howard Hughes Medical Institute Investigator. “CHARMs are an elegant solution to the problem of silencing disease genes, and they have the potential to have an important position in the future of genetic medicines.”

To treat a genetic disease, target the gene

Prion disease, which leads to swift neurodegeneration and death, is caused by the presence of misshapen versions of the prion protein. These cause a cascade effect in the brain: the faulty prion proteins deform other proteins, and together these proteins not only stop functioning properly but also form toxic aggregates that kill neurons. The most famous type of prion disease, known colloquially as mad cow disease, is infectious, but other forms of prion disease can occur spontaneously or be caused by faulty prion protein genes.

Most conventional drugs work by targeting a protein. CHARMs, however, work further upstream, turning off the gene that codes for the faulty protein so that the protein never gets made in the first place. CHARMs do this by epigenetic editing, in which a chemical tag gets added to DNA in order to turn off or silence a target gene. Unlike gene editing, epigenetic editing does not modify the underlying DNA — the gene itself remains intact. However, like gene editing, epigenetic editing is stable, meaning that a gene switched off by CHARM should remain off. This would mean patients would only have to take CHARM once, as opposed to protein-targeting medications that must be taken regularly as the cells’ protein levels replenish.

Research in animals suggests that the prion protein isn’t necessary in a healthy adult, and that in cases of disease, removing the protein improves or even eliminates disease symptoms. In a person who hasn’t yet developed symptoms, removing the protein should prevent disease altogether. In other words, epigenetic editing could be an effective approach for treating genetic diseases such as inherited prion diseases. The challenge is creating a new type of therapy.

Fortunately, the team had a good template for CHARM: a research tool called CRISPRoff that Weissman’s group previously developed for silencing genes. CRISPRoff uses building blocks from CRISPR gene editing technology, including the guide protein Cas9 that directs the tool to the target gene. CRISPRoff silences the targeted gene by adding methyl groups, chemical tags that prevent the gene from being transcribed, or read into RNA, and so from being expressed as protein. When the researchers tested CRISPRoff’s ability to silence the prion protein gene, they found that it was effective and stable.

Several of its properties, though, prevented CRISPRoff from being a good candidate for a therapy. The researchers’ goal was to create a tool based on CRISPRoff that was just as potent but also safe for use in humans, small enough to deliver to the brain, and designed to minimize the risk of silencing the wrong genes or causing side effects.

From research tool to drug candidate

Led by Neumann and Bertozzi, the researchers began engineering and applying their new epigenome editor. The first problem that they had to tackle was size, because the editor needs to be small enough to be packaged and delivered to specific cells in the body. Delivering genes into the human brain is challenging; many clinical trials have used adeno-associated viruses (AAVs) as gene-delivery vehicles, but these are small and can only contain a small amount of genetic code. CRISPRoff is way too big; the code for Cas9 alone takes up most of the available space.

The Weissman lab researchers decided to replace Cas9 with a much smaller zinc finger protein (ZFP). Like Cas9, ZFPs can serve as guide proteins to direct the tool to a target site in DNA. ZFPs are also common in human cells, meaning they are less likely to trigger an immune response against themselves than the bacterial Cas9.

Next, the researchers had to design the part of the tool that would silence the prion protein gene. At first, they used part of a methyltransferase, a molecule that adds methyl groups to DNA, called DNMT3A. However, in the particular configuration needed for the tool, the molecule was toxic to the cell. The researchers focused on a different solution: Instead of delivering outside DNMT3A as part of the therapy, the tool is able to recruit the cell’s own DNMT3A to the prion protein gene. This freed up precious space inside of the AAV vector and prevented toxicity.

The researchers also needed to activate DNMT3A. In the cell, DNMT3A is usually inactive until it interacts with certain partner molecules. This default inactivity prevents accidental methylation of genes that need to remain turned on. Neumann came up with an ingenious way around this by combining sections of DNMT3A’s partner molecules and connecting these to ZFPs that bring them to the prion protein gene. When the cell’s DNMT3A comes across this combination of parts, it activates, silencing the gene.

“From the perspectives of both toxicity and size, it made sense to recruit the machinery that the cell already has; it was a much simpler, more elegant solution,” Neumann says. “Cells are already using methyltransferases all of the time, and we’re essentially just tricking them into turning off a gene that they would normally leave turned on.”

Testing in mice showed that ZFP-guided CHARMs could eliminate more than 80 percent of the prion protein in the brain, while previous research has shown that as little as 21 percent elimination can improve symptoms.

Once the researchers knew that they had a potent gene silencer, they turned to the problem of off-target effects. The genetic code for a CHARM that gets delivered to a cell will keep producing copies of the CHARM indefinitely. However, after the prion protein gene is switched off, there is no benefit to this, only more time for side effects to develop, so they tweaked the tool so that after it turns off the prion protein gene, it then turns itself off.

Meanwhile, a complementary project from Broad Institute scientist and collaborator Benjamin Deverman’s lab, focused on brain-wide gene delivery and published in Science on May 17, has brought the CHARM technology one step closer to being ready for clinical trials. Although naturally occurring types of AAV have been used for gene therapy in humans before, they do not enter the adult brain efficiently, making it impossible to treat a whole-brain disease like prion disease. Tackling the delivery problem, Deverman’s group has designed an AAV vector that can get into the brain more efficiently by leveraging a pathway that naturally shuttles iron into the brain. Engineered vectors like this one make a therapy like CHARM one step closer to reality.

Thanks to these creative solutions, the researchers now have a highly effective epigenetic editor that is small enough to deliver to the brain, and that appears in cell culture and animal testing to have low toxicity and limited off-target effects.

“It’s been a privilege to be part of this; it’s pretty rare to go from basic research to therapeutic application in such a short amount of time,” Bertozzi says. “I think the key was forming a collaboration that took advantage of the Weissman lab’s tool-building experience, the Vallabh and Minikel lab’s deep knowledge of the disease, and the Deverman lab’s expertise in gene delivery.”

Looking ahead

With the major elements of the CHARM technology solved, the team is now fine-tuning their tool to make it more effective, safer, and easier to produce at scale, as will be necessary for clinical trials. They have already made the tool modular, so that its various pieces can be swapped out and future CHARMs won’t have to be programmed from scratch. CHARMs are also currently being tested as therapeutics in mice.

The path from basic research to clinical trials is a long and winding one, and the researchers know that CHARMs still have a way to go before they might become a viable medical option for people with prion diseases, including Vallabh, or other diseases with similar genetic components. However, with a strong therapy design and promising laboratory results in hand, the researchers have good reason to be hopeful. They continue to work at full throttle, intent on developing their technology so that it can save patients’ lives not someday, but as soon as possible.

With programmable pixels, novel sensor improves imaging of neural activity

New camera chip design allows for optimizing each pixel’s timing to maximize signal to noise ratio when tracking real-time visual indicator of neural voltage, described in a new paper from a team in the Wilson Lab published in Nature Communications.

David Orenstein | The Picower Institute for Learning and Memory
June 7, 2024
New technique reveals how gene transcription is coordinated in cells

By capturing short-lived RNA molecules, scientists can map relationships between genes and the regulatory elements that control them.

Anne Trafton | MIT News
June 5, 2024

The human genome contains about 23,000 genes, but only a fraction of those genes are turned on inside a cell at any given time. The complex network of regulatory elements that controls gene expression includes regions of the genome called enhancers, which are often located far from the genes that they regulate.

This distance can make it difficult to map the complex interactions between genes and enhancers. To overcome that, MIT researchers have invented a new technique that allows them to observe the timing of gene and enhancer activation in a cell. When a gene is turned on around the same time as a particular enhancer, it strongly suggests the enhancer is controlling that gene.

Learning more about which enhancers control which genes, in different types of cells, could help researchers identify potential drug targets for genetic disorders. Genomic studies have identified mutations in many non-protein-coding regions that are linked to a variety of diseases. Could these be unknown enhancers?

“When people start using genetic technology to identify regions of chromosomes that have disease information, most of those sites don’t correspond to genes. We suspect they correspond to these enhancers, which can be quite distant from a promoter, so it’s very important to be able to identify these enhancers,” says Phillip Sharp, an MIT Institute Professor Emeritus and member of MIT’s Koch Institute for Integrative Cancer Research.

Sharp is the senior author of the new study, which appears today in Nature. MIT Research Assistant D.B. Jay Mahat is the lead author of the paper.

Hunting for eRNA

Less than 2 percent of the human genome consists of protein-coding genes. The rest of the genome includes many elements that control when and how those genes are expressed. Enhancers, which are thought to turn genes on by coming into physical contact with gene promoter regions through transiently forming a complex, were discovered about 45 years ago.

More recently, in 2010, researchers discovered that these enhancers are transcribed into RNA molecules, known as enhancer RNA or eRNA. Scientists suspect that this transcription occurs when the enhancers are actively interacting with their target genes. This raised the possibility that measuring eRNA transcription levels could help researchers determine when an enhancer is active, as well as which genes it’s targeting.

“That information is extraordinarily important in understanding how development occurs, and in understanding how cancers change their regulatory programs and activate processes that lead to de-differentiation and metastatic growth,” Mahat says.

However, this kind of mapping has proven difficult to perform because eRNA is produced in very small quantities and does not last long in the cell. Additionally, eRNA lacks a modification known as a poly-A tail, which is the “hook” that most techniques use to pull RNA out of a cell.

One way to capture eRNA is to add a nucleotide to cells that halts transcription when incorporated into RNA. These nucleotides also contain a tag called biotin that can be used to fish the RNA out of a cell. However, this current technique only works on large pools of cells and doesn’t give information about individual cells.

While brainstorming ideas for new ways to capture eRNA, Mahat and Sharp considered using click chemistry, a technique that can be used to join two molecules together if they are each tagged with “click handles” that can react together.

The researchers designed nucleotides labeled with one click handle, and once these nucleotides are incorporated into growing eRNA strands, the strands can be fished out with a tag containing the complementary handle. This allowed the researchers to capture eRNA and then purify, amplify, and sequence it. Some RNA is lost at each step, but Mahat estimates that they can successfully pull out about 10 percent of the eRNA from a given cell.

Using this technique, the researchers obtained a snapshot of the enhancers and genes that are being actively transcribed at a given time in a cell.

“You want to be able to determine, in every cell, the activation of transcription from regulatory elements and from their corresponding gene. And this has to be done in a single cell because that’s where you can detect synchrony or asynchrony between regulatory elements and genes,” Mahat says.

Timing of gene expression

Demonstrating their technique in mouse embryonic stem cells, the researchers found that they could calculate approximately when a particular region starts to be transcribed, based on the length of the RNA strand and the speed of the polymerase (the enzyme responsible for transcription) — that is, how far the polymerase transcribes per second. This allowed them to determine which genes and enhancers were being transcribed around the same time.

The researchers used this approach to determine the timing of the expression of cell cycle genes in more detail than has previously been possible. They were also able to confirm several sets of known gene-enhancer pairs and generated a list of about 50,000 possible enhancer-gene pairs that they can now try to verify.

Learning which enhancers control which genes would prove valuable in developing new treatments for diseases with a genetic basis. Last year, the U.S. Food and Drug Administration approved the first gene therapy treatment for sickle cell anemia, which works by interfering with an enhancer that results in activation of a fetal globin gene, reducing the production of sickled blood cells.

The MIT team is now applying this approach to other types of cells, with a focus on autoimmune diseases. Working with researchers at Boston Children’s Hospital, they are exploring immune cell mutations that have been linked to lupus, many of which are found in non-coding regions of the genome.

“It’s not clear which genes are affected by these mutations, so we are beginning to tease apart the genes these putative enhancers might be regulating, and in what cell types these enhancers are active,” Mahat says. “This is a tool for creating gene-to-enhancer maps, which are fundamental in understanding the biology, and also a foundation for understanding disease.”

The findings of this study also offer evidence for a theory that Sharp has recently developed, along with MIT professors Richard Young and Arup Chakraborty, that gene transcription is controlled by membraneless droplets known as condensates. These condensates are made of large clusters of enzymes and RNA, which Sharp suggests may include eRNA produced at enhancer sites.

“We picture that the communication between an enhancer and a promoter is a condensate-type, transient structure, and RNA is part of that. This is an important piece of work in building the understanding of how RNAs from enhancers could be active,” he says.

The research was funded by the National Cancer Institute, the National Institutes of Health, and the Emerald Foundation Postdoctoral Transition Award.