Research Area: Computational Biology

Establishing boundaries of the genetic kind

The pseudoautosomal region (PAR) is a critical area on the Y chromosome that swaps genetic information with the X chromosome. Recent research from the Page Lab reaffirms the location of PAR and offers a refined understanding of where crossover events occur.

Shafaq Zia | Whitehead Institute
October 14, 2024

At first, the X and the Y sex chromosomes seemed like an unlikely pair. But then, researchers, including Whitehead Institute Member David Page, began finding clues that suggested otherwise: identical DNA sequences on the X and Y chromosomes.

Soon, it became clear that the tips of the X and Y chromosomes join together in a tight embrace, swapping genetic material during the process of sperm production from immature male germ cells. This limited area of genetic exchange between the two sex chromosomes is called the pseudoautosomal region (PAR).

But science is an iterative process—a continuous cycle of questioning, testing, and revising knowledge. Last fall, what had long been considered well established in genetics was called into question when new research suggested that the boundary of the PAR might be half a million base pairs away from the accepted location. Given that a typical human gene is about tens of thousands of base pairs, this length would potentially span multiple genes on the X and Y chromosomes, raising serious concerns about the accuracy and validity of decades of scientific literature.

Fortunately, new work from Page, research scientist Daniel Winston Bellott, and colleagues—published Oct. 14 in the American Journal of Human Genetics—offers clarity. In this study, the group re-examines the size of the PAR using sequencing data presented by outside researchers in their 2023 work, alongside decades of genomic resources, and single-cell sequencing of human sperm. Their findings confirm that the location of the boundary to the PAR, as identified by scientists in 1989, still holds true.

“If one is interested in understanding sex differences in health and disease, the boundary of the pseudoautosomal region is arguably the most fundamental landmark in the genome,” says Page, who is also a professor of biology at the Massachusetts Institute of Technology and an Investigator with Howard Hughes Medical Institute. “Had this boundary been multiple genes off, the field would have been shaken to its foundations.”

Dance of the chromosomes

The X and Y chromosomes evolved from an ancestral pair of chromosomes with identical structures. Over time, the Y chromosome degenerated drastically, losing hundreds of functional genes. Despite their differences, the X and Y chromosomes come together during a special type of cell division called male meiosis, which produces sperm cells.

This process begins with the tips of the sex chromosomes aligning side by side like two strands of rope. As the X and Y chromosomes embrace each other, enzymes create breaks in the DNA. These breaks are repaired using the opposite chromosome as a template, linking the X and Y together. About half of the time, an entire segment of DNA, which often contains multiple genes, will cross over onto the opposite chromosome.

The genetic exchange, called recombination, concludes with the X and Y chromosomes being pulled apart to opposite ends of the dividing cell, ensuring that each chromosome ends up in a different daughter cell. “This intricate dance of the X and Y chromosomes is essential to a sperm getting either an X or a Y—not both, and not neither,” says Page.

This way when the sperm—carrying either an X or a Y—fuses with the egg—carrying an X—during fertilization, the resulting zygote has the right number of chromosomes and a mix of genetic material from both parents.

But that’s not all. The swapping of DNA during recombination also allows for the chromosomes to have the same genes but with slight variations. These unique combinations of genetic material across sex chromosomes are key to genetic diversity within a species, enabling it to survive, adapt, and reproduce successfully.

Beyond the region of recombination, the Y chromosome contains genes that are important for sex determination, for sperm production, and for general cellular functioning. The primary sex-defining gene, SRY, which triggers the development of an embryo into a male, is located only 10,000 bases from the boundary of the PAR.

Advancing together

To determine whether the location of this critical boundary on the human sex chromosomes—where they stop crossing over during meiosis and become X-specific or Y-specific—had been misidentified for over three decades, researchers began by comparing publicly-available DNA sequences from the X and the Y chromosomes of seven primate species: humans, chimpanzees, gorillas, orangutans, siamangs, rhesus macaques, and colobus monkeys.

Based on the patterns of crossover between the X and the Y chromosomes of these species, the researchers constructed an evolutionary tree. Upon analyzing how DNA sequences close to and distant from the PAR boundary group together across species, the researchers found a substitution mutation—where a letter in a long string of letters is swapped for a different one—in the DNA of the human X and Y chromosomes. This change was also present in the chimpanzee Y chromosome, suggesting that the mutation originally occurred in the last common ancestor of humans and chimpanzees and was then transferred to the human X chromosome.

“These alignments between various primates allowed us to observe where the X and the Y chromosomes have preserved identity over millions of years and where they have diverged,” says Bellott. “That [pseudoautosomal] boundary has remained unchanged for 25 million years.”

Next, the group studied crossover events in living humans using a vast dataset of single-cell sequencing of sperm samples. They found 795 sperm with clear swapping of genetic material somewhere between the originally proposed boundary of the PAR and the newly-proposed 2023 boundary.

Once these analyses confirmed that the original location of the PAR boundary remains valid, Page and his team turned their attention to data from the 2023 study that contested this 1989 finding. The researchers focused on 10 male genomes assembled by the outside group, which contained contiguous sequences from the PAR.

Since substitutions on the Y chromosome typically occur at a steady rate, but in the PAR, changes on the X chromosome can transfer to the Y through recombination, the researchers compared the DNA sequences from the ten genomes to determine whether they followed the expected steady rate of change or if they varied.

The team found that close to the originally proposed PAR boundary, the DNA sequences changed at a steady rate. But further away from the boundary, the rate of change varied, suggesting that crossover events likely occurred in this region. Furthermore, the group identified several shared genetic differences between the X and the Y chromosomes of these genomes, which demonstrates that recombination has occurred even closer to the PAR boundary than scientists observed in 1989.

“Ironically, instead of contradicting the original boundary, the 2023 work has helped us refine the location of crossover to an even narrower area near the boundary,” says Page.

Thanks to the efforts of Page’s group at Whitehead Institute, our understanding of the PAR is clearer than ever, and business can go on as usual for researchers investigating sex differences in health and disease.

Cancer biologists discover a new mechanism for an old drug

Study reveals the drug, 5-fluorouracil, acts differently in different types of cancer — a finding that could help researchers design better drug combinations.

Anne Trafton | MIT News
October 7, 2024

Since the 1950s, a chemotherapy drug known as 5-fluorouracil has been used to treat many types of cancer, including blood cancers and cancers of the digestive tract.

Doctors have long believed that this drug works by damaging the building blocks of DNA. However, a new study from MIT has found that in cancers of the colon and other gastrointestinal cancers, it actually kills cells by interfering with RNA synthesis.

The findings could have a significant effect on how doctors treat many cancer patients. Usually, 5-fluorouracil is given in combination with chemotherapy drugs that damage DNA, but the new study found that for colon cancer, this combination does not achieve the synergistic effects that were hoped for. Instead, combining 5-FU with drugs that affect RNA synthesis could make it more effective in patients with GI cancers, the researchers say.

“Our work is the most definitive study to date showing that RNA incorporation of the drug, leading to an RNA damage response, is responsible for how the drug works in GI cancers,” says Michael Yaffe, a David H. Koch Professor of Science at MIT, the director of the MIT Center for Precision Cancer Medicine, and a member of MIT’s Koch Institute for Integrative Cancer Research. “Textbooks implicate the DNA effects of the drug as the mechanism in all cancer types, but our data shows that RNA damage is what’s really important for the types of tumors, like GI cancers, where the drug is used clinically.”

Yaffe, the senior author of the new study, hopes to plan clinical trials of 5-fluorouracil with drugs that would enhance its RNA-damaging effects and kill cancer cells more effectively.

Jung-Kuei Chen, a Koch Institute research scientist, and Karl Merrick, a former MIT postdoc, are the lead authors of the paper, which appears today in Cell Reports Medicine.

An unexpected mechanism

Clinicians use 5-fluorouracil (5-FU) as a first-line drug for colon, rectal, and pancreatic cancers. It’s usually given in combination with oxaliplatin or irinotecan, which damage DNA in cancer cells. The combination was thought to be effective because 5-FU can disrupt the synthesis of DNA nucleotides. Without those building blocks, cells with damaged DNA wouldn’t be able to efficiently repair the damage and would undergo cell death.

Yaffe’s lab, which studies cell signaling pathways, wanted to further explore the underlying mechanisms of how these drug combinations preferentially kill cancer cells.

The researchers began by testing 5-FU in combination with oxaliplatin or irinotecan in colon cancer cells grown in the lab. To their surprise, they found that not only were the drugs not synergistic, in many cases they were less effective at killing cancer cells than what one would expect by simply adding together the effects of 5-FU or the DNA-damaging drug given alone.

“One would have expected that these combinations to cause synergistic cancer cell death because you are targeting two different aspects of a shared process: breaking DNA, and making nucleotides,” Yaffe says. “Karl looked at a dozen colon cancer cell lines, and not only were the drugs not synergistic, in most cases they were antagonistic. One drug seemed to be undoing what the other drug was doing.”

Yaffe’s lab then teamed up with Adam Palmer, an assistant professor of pharmacology at the University of North Carolina School of Medicine, who specializes in analyzing data from clinical trials. Palmer’s research group examined data from colon cancer patients who had been on one or more of these drugs and showed that the drugs did not show synergistic effects on survival in most patients.

“This confirmed that when you give these combinations to people, it’s not generally true that the drugs are actually working together in a beneficial way within an individual patient,” Yaffe says. “Instead, it appears that one drug in the combination works well for some patients while another drug in the combination works well in other patients. We just cannot yet predict which drug by itself is best for which patient, so everyone gets the combination.”

These results led the researchers to wonder just how 5-FU was working, if not by disrupting DNA repair. Studies in yeast and mammalian cells had shown that the drug also gets incorporated into RNA nucleotides, but there has been dispute over how much this RNA damage contributes to the drug’s toxic effects on cancer cells.

Inside cells, 5-FU is broken down into two different metabolites. One of these gets incorporated into DNA nucleotides, and other into RNA nucleotides. In studies of colon cancer cells, the researchers found that the metabolite that interferes with RNA was much more effective at killing colon cancer cells than the one that disrupts DNA.

That RNA damage appears to primarily affect ribosomal RNA, a molecule that forms part of the ribosome — a cell organelle responsible for assembling new proteins. If cells can’t form new ribosomes, they can’t produce enough proteins to function. Additionally, the lack of undamaged ribosomal RNA causes cells to destroy a large set of proteins that normally bind up the RNA to make new functional ribosomes.

The researchers are now exploring how this ribosomal RNA damage leads cells to under programmed cell death, or apoptosis. They hypothesize that sensing of the damaged RNAs within cell structures called lysosomes somehow triggers an apoptotic signal.

“My lab is very interested in trying to understand the signaling events during disruption of ribosome biogenesis, particularly in GI cancers and even some ovarian cancers, that cause the cells to die. Somehow, they must be monitoring the quality control of new ribosome synthesis, which somehow is connected to the death pathway machinery,” Yaffe says.

New combinations

The findings suggest that drugs that stimulate ribosome production could work together with 5-FU to make a highly synergistic combination. In their study, the researchers showed that a molecule that inhibits KDM2A, a suppressor of ribosome production, helped to boost the rate of cell death in colon cancer cells treated with 5-FU.

The findings also suggest a possible explanation for why combining 5-FU with a DNA-damaging drug often makes both drugs less effective. Some DNA damaging drugs send a signal to the cell to stop making new ribosomes, which would negate 5-FU’s effect on RNA. A better approach may be to give each drug a few days apart, which would give patients the potential benefits of each drug, without having them cancel each other out.

“Importantly, our data doesn’t say that these combination therapies are wrong. We know they’re effective clinically. It just says that if you adjust how you give these drugs, you could potentially make those therapies even better, with relatively minor changes in the timing of when the drugs are given,” Yaffe says.

He is now hoping to work with collaborators at other institutions to run a phase 2 or 3 clinical trial in which patients receive the drugs on an altered schedule.

“A trial is clearly needed to look for efficacy, but it should be straightforward to initiate because these are already clinically accepted drugs that form the standard of care for GI cancers. All we’re doing is changing the timing with which we give them,” he says.

The researchers also hope that their work could lead to the identification of biomarkers that predict which patients’ tumors will be more susceptible to drug combinations that include 5-FU. One such biomarker could be RNA polymerase I, which is active when cells are producing a lot of ribosomal RNA.

The research was funded by the Damon Runyon Cancer Research Fund, a Ludwig Center at MIT Fellowship, the National Institutes of Health, the Ovarian Cancer Research Fund, the Holloway Foundation, and the STARR Cancer Consortium.

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.”

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.”

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.

“Rosetta Stone” of cell signaling could expedite precision cancer medicine

An atlas of human protein kinases enables scientists to map cell signaling pathways with unprecedented speed and detail. Michael Yaffe, the David H. Koch Professor of Science at MIT, the director of the MIT Center for Precision Cancer Medicine, a member of MIT’s Koch Institute for Integrative Cancer Research, and a senior author of the new study published in Nature, is hoping to apply the comprehensive atlas of enzymes that regulate a wide variety of cellular activities to individual patients’ tumors to learn more about how the signaling states differ in cancer cancer, which could reveal new

Megan Scudellari | Koch Institute
June 3, 2024

A newly complete database of human protein kinases and their preferred binding sites provides a powerful new platform to investigate cell signaling pathways.

Culminating 25 years of research, MIT, Harvard University, and Yale University scientists and collaborators have unveiled a comprehensive atlas of human tyrosine kinases — enzymes that regulate a wide variety of cellular activities — and their binding sites.

The addition of tyrosine kinases to a previously published dataset from the same group now completes a free, publicly available atlas of all human kinases and their specific binding sites on proteins, which together orchestrate fundamental cell processes such as growth, cell division, and metabolism.

Now, researchers can use data from mass spectrometry, a common laboratory technique, to identify the kinases involved in normal and dysregulated cell signaling in human tissue, such as during inflammation or cancer progression.

“I am most excited about being able to apply this to individual patients’ tumors and learn about the signaling states of cancer and heterogeneity of that signaling,” says Michael Yaffe, who is the David H. Koch Professor of Science at MIT, the director of the MIT Center for Precision Cancer Medicine, a member of MIT’s Koch Institute for Integrative Cancer Research, and a senior author of the new study. “This could reveal new druggable targets or novel combination therapies.”

The study, published in Nature, is the product of a long-standing collaboration with senior authors Lewis Cantley at Harvard Medical School and Dana-Farber Cancer Institute, Benjamin Turk at Yale School of Medicine, and Jared Johnson at Weill Cornell Medical College.

The paper’s lead authors are Tomer Yaron-Barir at Columbia University Irving Medical Center, and MIT’s Brian Joughin, with contributions from Kontstantin Krismer, Mina Takegami, and Pau Creixell.

Kinase kingdom

Human cells are governed by a network of diverse protein kinases that alter the properties of other proteins by adding or removing chemical compounds called phosphate groups. Phosphate groups are small but powerful: When attached to proteins, they can turn proteins on or off, or even dramatically change their function. Identifying which of the almost 400 human kinases phosphorylate a specific protein at a particular site on the protein was traditionally a lengthy, laborious process.

Beginning in the mid 1990s, the Cantley laboratory developed a method using a library of small peptides to identify the optimal amino acid sequence — called a motif, similar to a scannable barcode — that a kinase targets on its substrate proteins for the addition of a phosphate group. Over the ensuing years, Yaffe, Turk, and Johnson, all of whom spent time as postdocs in the Cantley lab, made seminal advancements in the technique, increasing its throughput, accuracy, and utility.

Johnson led a massive experimental effort exposing batches of kinases to these peptide libraries and observed which kinases phosphorylated which subsets of peptides. In a corresponding Nature paper published in January 2023, the team mapped more than 300 serine/threonine kinases, the other main type of protein kinase, to their motifs. In the current paper, they complete the human “kinome” by successfully mapping 93 tyrosine kinases to their corresponding motifs.

Next, by creating and using advanced computational tools, Yaron-Barir, Krismer, Joughin, Takegami, and Yaffe tested whether the results were predictive of real proteins, and whether the results might reveal unknown signaling events in normal and cancer cells. By analyzing phosphoproteomic data from mass spectrometry to reveal phosphorylation patterns in cells, their atlas accurately predicted tyrosine kinase activity in previously studied cell signaling pathways.

For example, using recently published phosphoproteomic data of human lung cancer cells treated with two targeted drugs, the atlas identified that treatment with erlotinib, a known inhibitor of the protein EGFR, downregulated sites matching a motif for EGFR. Treatment with afatinib, a known HER2 inhibitor, downregulated sites matching the HER2 motif. Unexpectedly, afatinib treatment also upregulated the motif for the tyrosine kinase MET, a finding that helps explain patient data linking MET activity to afatinib drug resistance.

Actionable results

There are two key ways researchers can use the new atlas. First, for a protein of interest that is being phosphorylated, the atlas can be used to narrow down hundreds of kinases to a short list of candidates likely to be involved. “The predictions that come from using this will still need to be validated experimentally, but it’s a huge step forward in making clear predictions that can be tested,” says Yaffe.

Second, the atlas makes phosphoproteomic data more useful and actionable. In the past, researchers might gather phosphoproteomic data from a tissue sample, but it was difficult to know what that data was saying or how to best use it to guide next steps in research. Now, that data can be used to predict which kinases are upregulated or downregulated and therefore which cellular signaling pathways are active or not.

“We now have a new tool now to interpret those large datasets, a Rosetta Stone for phosphoproteomics,” says Yaffe. “It is going to be particularly helpful for turning this type of disease data into actionable items.”

In the context of cancer, phosophoproteomic data from a patient’s tumor biopsy could be used to help doctors quickly identify which kinases and cell signaling pathways are involved in cancer expansion or drug resistance, then use that knowledge to target those pathways with appropriate drug therapy or combination therapy.

Yaffe’s lab and their colleagues at the National Institutes of Health are now using the atlas to seek out new insights into difficult cancers, including appendiceal cancer and neuroendocrine tumors. While many cancers have been shown to have a strong genetic component, such as the genes BRCA1 and BRCA2 in breast cancer, other cancers are not associated with any known genetic cause. “We’re using this atlas to interrogate these tumors that don’t seem to have a clear genetic driver to see if we can identify kinases that are driving cancer progression,” he says.

Biological insights

In addition to completing the human kinase atlas, the team made two biological discoveries in their recent study. First, they identified three main classes of phosphorylation motifs, or barcodes, for tyrosine kinases. The first class is motifs that map to multiple kinases, suggesting that numerous signaling pathways converge to phosphorylate a protein boasting that motif. The second class is motifs with a one-to-one match between motif and kinase, in which only a specific kinase will activate a protein with that motif. This came as a partial surprise, as tyrosine kinases have been thought to have minimal specificity by some in the field.

The final class includes motifs for which there is no clear match to one of the 78 classical tyrosine kinases. This class includes motifs that match to 15 atypical tyrosine kinases known to also phosphorylate serine or threonine residues. “This means that there’s a subset of kinases that we didn’t recognize that are actually playing an important role,” says Yaffe. It also indicates there may be other mechanisms besides motifs alone that affect how a kinase interacts with a protein.

The team also discovered that tyrosine kinase motifs are tightly conserved between humans and the worm species C. elegans, despite the species being separated by more than 600 million years of evolution. In other words, a worm kinase and its human homologue are phosphorylating essentially the same motif. That sequence preservation suggests that tyrosine kinases are highly critical to signaling pathways in all multicellular organisms, and any small change would be harmful to an organism.

The research was funded by the Charles and Marjorie Holloway Foundation, the MIT Center for Precision Cancer Medicine, the Koch Institute Frontier Research Program via L. Scott Ritterbush, the Leukemia and Lymphoma Society, the National Institutes of Health, Cancer Research UK, the Brain Tumour Charity, and the Koch Institute Support (core) grant from the National Cancer Institute.

Taking RNAi from interesting science to impactful new treatments

Alnylam Pharmaceuticals is translating the promise of RNA interference (RNAi) research into a new class of powerful, gene-based therapies. These days Alnylam is not the only company developing RNAi-based medicines, but it is still a pioneer in the field. The company’s founders — MIT Institute Professor Phil Sharp, Professor David Bartel, Professor Emeritus Paul Schimmel, and former MIT postdocs Thomas Tuschl and Phillip Zamore — see Alnylam as a champion for the field more broadly.

Zach Winn | MIT News
May 13, 2024

There are many hurdles to clear before a research discovery becomes a life-changing treatment for patients. That’s especially true when the treatments being developed represent an entirely new class of medicines. But overcoming those obstacles can revolutionize our ability to treat diseases.

Few companies exemplify that process better than Alnylam Pharmaceuticals. Alnylam was founded by a group of MIT-affiliated researchers who believed in the promise of a technology — RNA interference, or RNAi.

The researchers had done foundational work to understand how RNAi, which is a naturally occurring process, works to silence genes through the degradation of messenger RNA. But it was their decision to found Alnylam in 2002 that attracted the funding and expertise necessary to turn their discoveries into a new class of medicines. Since that decision, Alnylam has made remarkable progress taking RNAi from an interesting scientific discovery to an impactful new treatment pathway.

Today Alnylam has five medicines approved by the U.S. Food and Drug Administration (one Alnylam-discovered RNAi therapeutic is licensed to Novartis) and a rapidly expanding clinical pipeline. The company’s approved medicines are for debilitating, sometimes fatal conditions that many patients have grappled with for decades with few other options.

The company estimates its treatments helped more than 5,000 patients in 2023 alone. Behind that number are patient stories that illustrate how Alnylam has changed lives. A mother of three says Alnylam’s treatments helped her take back control of her life after being bed-ridden with attacks associated with the rare genetic disease acute intermittent porphyria (AIP). Another patient reported that one of the company’s treatments helped her attend her daughter’s wedding. A third patient, who had left college due to frequent AIP attacks, was able to return to school.

These days Alnylam is not the only company developing RNAi-based medicines. But it is still a pioneer in the field, and the company’s founders — MIT Institute Professor Phil Sharp, Professor David Bartel, Professor Emeritus Paul Schimmel, and former MIT postdocs Thomas Tuschl and Phillip Zamore — see Alnylam as a champion for the field more broadly.

“Alnylam has published more than 250 scientific papers over 20 years,” says Sharp, who currently serves as chair of Alnylam’s scientific advisory board. “Not only did we do the science, not only did we translate it to benefit patients, but we also described every step. We established this as a modality to treat patients, and I’m very proud of that record.”

Pioneering RNAi development

MIT’s involvement in RNAi dates back to its discovery. Before Andrew Fire PhD ’83 shared a Nobel Prize for the discovery of RNAi in 1998, he worked on understanding how DNA was transcribed into RNA, as a graduate student in Sharp’s lab.

After leaving MIT, Fire and collaborators showed that double-stranded RNA could be used to silence specific genes in worms. But the biochemical mechanisms that allowed double-stranded RNA to work were unknown until MIT professors Sharp, Bartel, and Ruth Lehmann, along with Zamore and Tuschl, published foundational papers explaining the process. The researchers developed a system for studying RNAi and showed how RNAi can be controlled using different genetic sequences. Soon after Tuschl left MIT, he showed that a similar process could also be used to silence specific genes in human cells, opening up a new frontier in studying genes and ultimately treating diseases.

“Tom showed you could synthesize these small RNAs, transfect them into cells, and get a very specific knockdown of the gene that corresponded to that the small RNAs,” Bartel explains. “That discovery transformed biological research. The ability to specifically knockdown a mammalian gene was huge. You could suddenly study the function of any gene you were interested in by knocking it down and seeing what happens. … The research community immediately started using that approach to study the function of their favorite genes in mammalian cells.”

Beyond illuminating gene function, another application came to mind.

“Because almost all diseases are related to genes, could we take these small RNAs and silence genes to treat patients?” Sharp remembers wondering.

To answer the question, the researchers founded Alnylam in 2002. (They recruited Schimmel, a biotech veteran, around the same time.) But there was a lot of work to be done before the technology could be tried in patients. The main challenge was getting RNAi into the cytoplasm of the patients’ cells.

“Through work in Dave Bartel and Phil Sharp’s lab, among others, it became evident that to make RNAi into therapies, there were three problems to solve: delivery, delivery, and delivery,” says Alnylam Chief Scientific Officer Kevin Fitzgerald, who has been with the company since 2005.

Early on, Alnylam collaborated with MIT drug delivery expert and Institute Professor Bob Langer. Eventually, Alnylam developed the first lipid nanoparticles (LNPs) that could be used to encase RNA and deliver it into patient cells. LNPs were later used in the mRNA vaccines for Covid-19.

“Alnylam has invested over 20 years and more than $4 billion in RNAi to develop these new therapeutics,” Sharp says. “That is the means by which innovations can be translated to the benefit of society.”

From scientific breakthrough to patient bedside

Alnylam received its first FDA approval in 2018 for treatment of the polyneuropathy of hereditary transthyretin-mediated amyloidosis, a rare and fatal disease. It doubled as the first RNAi therapeutic to reach the market and the first drug approved to treat that condition in the United States.

“What I keep in mind is, at the end of the day for certain patients, two months is everything,” Fitzgerald says. “The diseases that we’re trying to treat progress month by month, day by day, and patients can get to a point where nothing is helping them. If you can move their disease by a stage, that’s huge.”

Since that first treatment, Alnylam has updated its RNAi delivery system — including by conjugating small interfering RNAs to molecules that help them gain entry to cells — and earned approvals to treat other rare genetic diseases along with high cholesterol (the treatment licensed to Novartis). All of those treatments primarily work by silencing genes that encode for the production of proteins in the liver, which has proven to be the easiest place to deliver RNAi molecules. But Alnylam’s team is confident they can deliver RNAi to other areas of the body, which would unlock a new world of treatment possibilities. The company has reported promising early results in the central nervous system and says a phase one study last year was the first RNAi therapeutic to demonstrate gene silencing in the human brain.

“There’s a lot of work being done at Alnylam and other companies to deliver these RNAis to other tissues: muscles, immune cells, lung cells, etc.,” Sharp says. “But to me the most interesting application is delivery to the brain. We think we have a therapeutic modality that can very specifically control the activity of certain genes in the nervous system. I think that’s extraordinarily important, for diseases from Alzheimer’s to schizophrenia and depression.”

The central nervous system work is particularly significant for Fitzgerald, who watched his father struggle with Parkinson’s.

“Our goal is to be in every organ in the human body, and then combinations of organs, and then combinations of targets within individual organs, and then combinations of targets within multi-organs,” Fitzgerald says. “We’re really at the very beginning of what this technology is going do for human health.”

It’s an exciting time for the RNAi scientific community, including many who continue to study it at MIT. Still, Alnylam will need to continue executing in its drug development efforts to deliver on that promise and help an expanding pool of patients.

“I think this is a real frontier,” Sharp says. “There’s major therapeutic need, and I think this technology could have a huge impact. But we have to prove it. That’s why Alnylam exists: to pursue new science that unlocks new possibilities and discover if they can be made to work. That, of course, also why MIT is here: to improve lives.”

Q&A: Pulin Li on recreating development in the lab

In the whirlwind of activity that occurs simultaneously in a developing embryo, it can be difficult for scientists to pinpoint critical moments of a particular trait. In this Q&A, Pulin Li discusses how her lab ventures beyond mere observation to actually engineer developmental events in a petri dish, and why this approach is vital for understanding health and disease more broadly.

Shafaq Zia | Whitehead Institute
May 1, 2024
Uncovering answers to longstanding questions about sex differences in autoimmune and neurodegenerative diseases

Whitehead Institute researchers including those in the Page Lab and Corradin Lab are investigating the role of X and Y chromosomes beyond sex determination, paying close attention to conditions that mostly — or distinctly — affect females, and mentoring the next generation of researchers to challenge the status quo for a better world.

Shafaq Zia | Whitehead Institute
March 12, 2024