Research Area: Biochemistry, Biophysics, and Structural Biology

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.

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.

“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.”

De-tail-ing RNA regulation in eggs and early embryos

For a brief period during embryonic development, cells must rely on messenger RNAs provided by the maternal genome. In Developmental Cell, Bartel Lab members detail how cells regulate this limited supply of genetic material.

Greta Friar | Whitehead Institute
March 6, 2024
News brief: Davis Lab

Exploring the cellular neighborhood

Alison Biester | Department of Biology
March 12, 2024

New software allows scientists to model shapeshifting proteins in native cellular environments

Cells rely on complex molecular machines composed of protein assemblies to perform essential functions such as energy production, gene expression, and protein synthesis. To better understand how these machines work, scientists capture snapshots of them by isolating proteins from cells and using various methods to determine their structures. However, isolating proteins from cells also removes them from the context of their native environment, including protein interaction partners and cellular location.

Recently, cryogenic electron tomography (cryo-ET) has emerged as a way to observe proteins in their native environment by imaging frozen cells at different angles to obtain three-dimensional structural information. This approach is exciting because it allows researchers to directly observe how and where proteins associate with each other, revealing the cellular neighborhood of those interactions within the cell.

With the technology available to image proteins in their native environment, graduate student Barrett Powell wondered if he could take it one step further: what if molecular machines could be observed in action? In a paper published today in Nature Methods, Powell describes the method he developed, called tomoDRGN, for modeling structural differences of proteins in cryo-ET data that arise from protein motions or proteins binding to different interaction partners. These variations are known as structural heterogeneity. 

Although Powell had joined the Davis Lab as an experimental scientist, he recognized the potential impact of computational approaches in understanding structural heterogeneity within a cell. Previously, the Davis Lab developed a related methodology named cryoDRGN to understand structural heterogeneity in purified samples. As Powell and Associate Professor of Biology Joey Davis saw cryo-ET rising in prominence in the field, Powell took on the challenge of reimagining this framework to work in cells. 

When solving structures with purified samples, each particle is imaged only once. By contrast, cryo-ET data is collected by imaging each particle more than 40 times from different angles. That meant tomoDRGN needed to be able to merge the information from more than 40 images, which was where the project hit a roadblock: the amount of data led to an information overload.

To address the information overload, Powell successfully rebuilt the cryoDRGN model to prioritize only the highest-quality data. When imaging the same particle multiple times, radiation damage occurs. The images acquired earlier, therefore, tend to be of higher quality because the particles are less damaged.

“By excluding some of the lower quality data, the results were actually better than using all of the data–and the computational performance was substantially faster,” Powell says.

Just as Powell was beginning work on testing his model, he had a stroke of luck: the authors of a groundbreaking new study that visualized, for the first time, ribosomes inside cells at near-atomic resolution, shared their raw data on the Electric Microscopy Public Image Archive (EMPIAR). This dataset was an exemplary test case for Powell, through which he demonstrated that tomoDRGN could uncover structural heterogeneity within cryo-ET data. 

According to Powell, one exciting result is what tomoDRGN found surrounding a subset of ribosomes in the EMPIAR dataset. Some of the ribosomal particles were associated with a bacterial cell membrane and engaged in a process called cotranslational translocation. This occurs when a protein is being simultaneously synthesized and transported across a membrane. Researchers can use this result to make new hypotheses about how the ribosome functions with other protein machinery integral to transporting proteins outside of the cell, now guided by a structure of the complex in its native environment. 

After seeing that tomoDRGN could resolve structural heterogeneity from a structurally diverse dataset, Powell was curious: how small of a population could tomoDRGN identify? For that test, he chose a protein named apoferritin which is a commonly used benchmark for cryo-ET and is often treated as structurally homogeneous. Ferritin is a protein used for iron storage and is referred to as apoferritin when it lacks iron.

Surprisingly, in addition to the expected particles, tomoDRGN revealed a minor population of ferritin particles–with iron bound–making up just 2% of the dataset that was not previously reported. This result further demonstrated tomoDRGN’s ability to identify structural states that occur so infrequently that they would be averaged out with traditional analysis tools. 

Powell and other members of the Davis Lab are excited to see how tomoDRGN can be applied to further ribosomal studies and to other systems. Davis works on understanding how cells assemble, regulate, and degrade molecular machines, so the next steps include exploring ribosome biogenesis within cells in greater detail using this new tool.

“What are the possible states that we may be losing during purification?” Davis says. “Perhaps more excitingly, we can look at how they localize within the cell and what partners and protein complexes they may be interacting with.” 

How signaling proteins get to the mitochondrial surface

Whitehead Institute Member Jonathan Weissman and colleagues used large-scale systematic genetic screens to identify the molecules and pathways that populate the mitochondrial surface with important and diverse signaling proteins. They deciphered the logic by which the cell ensures the proper delivery of these proteins. These findings may have important implications for understanding the impact on health and disease when these processes go awry.

Greta Friar | Whitehead Institute
February 26, 2024
News Brief: Vos Lab

Poise or Pause: researchers expand understanding of transcription factor’s role with newly discovered conformation

Lillian Eden | Department of Biology
February 23, 2024

New research from the Vos Lab in the Department of Biology at MIT reveals the dynamic nature of elongation factor protein key for regulating early stages of gene expression.

Transcription, the process of copying RNA from DNA, is a critical first step for cells to create proteins. The enzyme responsible for transcription is a motor protein called RNA polymerase. 

When an RNA polymerase transcribes a gene, it will begin elongating the mRNA and will then, often, pause. 

From there, the RNA polymerase will either return to elongating the mRNA or it will get stuck. For the latter occurrence, the mRNA and subsequent protein will never be made: the polymerase will go somewhere else, or restart transcription on the same gene and get stuck again. 

Pausing is thought to be governed by a protein called Negative Elongation Factor, NELF, and DRB-sensitivity inducing factor, DSIF. Previous research suggested that NELF stably clamps down onto RNA polymerase to stall the elongation process and prevent the polymerase from moving. That model contradicted cell-based experiments, however, which indicated that NELF is somehow still attached to polymerase after transcription resumes. 

New research from the Vos Lab in the Department of Biology at MIT published today in Molecular Cell reveals that NELF isn’t merely an on-off switch for transcription. Instead, NELF can change into a distinct conformation that allows the polymerase to resume transcription. The researchers dubbed this distinct conformation NELF’s “poised” state.

RNA polymerase pausing, sometimes for minutes at a time, is thought to be an important gene expression checkpoint; more than half of genes exhibit pausing, although many questions remain about the role of pausing in gene expression. Understanding both how and why the process is occurring, down to the atomic level, and what components are involved, is key to understanding how cells function, both individually and as part of an organism.

“It’s a very central question to biological research, and we still don’t fully understand it because it’s such a complex process,” says first author Bonnie G. Su, a graduate student in the Vos lab. “The bigger picture is: how does the cell decide what resources to allocate to certain biological processes? This finding might help us answer questions like that.” 

To visualize the two distinct conformations of NELF and polymerase, the researchers used a combination of biochemical and structural approaches. The previous understanding of proximal pausing was based on Cryo-Electron Microscopy images of the static complex. Cryo-EM is a powerful microscopy technique that involves freezing samples and imaging them, and that approach had captured polymerase in its paused state. 

Using the core Cryo-EM facility available at MIT.nano, Su instead added the necessary components for the polymerase to transcribe, and gathered structural data on an actively transcribing complex —allowing, for the first time, a stepwise visualization of how proximal pausing occurs. 

“What we found is that NELF, which we always thought of as static, can actually move around,” Su says. “This has updated our understanding of what pausing is, and how early gene regulation happens.” 

The structural results also provide an explanation for how polymerase may be cycling between the two states—and how one form of NELF may be forcing polymerase to pause, while the newly discovered form allows polymerase to resume transcription. 

It’s still unclear what triggers NELF to transition from the paused state to the poised state, and many questions remain about how polymerase is regulated, according to senior author Seychelle Vos, the Robert A. Swanson (1969) Career Development Professor of Life Sciences and HHMI Freeman Hrabowski Scholar. RNA polymerase can be associated with and is known to be regulated by a large repertoire of proteins. 

“We’re trying to see if we can actually lock the complex in the paused state by adding additional factors,” Vos says. “We’re also pursuing whether sequence context is affecting pausing behavior—how or if the sequence of DNA may be causing polymerase to pause.”

Protein production glitches in Huntington’s disease revealed

Research from the Jain Lab finds that, in Huntington's disease, repeats of certain nucleotides too many times in a row interferes with splicing.

January 30, 2024
Capsid of HIV-1 behaves like cell’s cargo receptor to enter the nucleus

Biologists demonstrate that HIV-1 capsid acts like a Trojan horse to pass viral cargo across the nuclear pore.

Lillian Eden | Department of Biology
January 24, 2024

Retroviruses cannot replicate on their own — they must insert their genetic code into the DNA of a host and exploit the host cell’s resources to make more copies of themselves, furthering infection. Some retroviruses only infect cells as they divide, when the nuclear envelope that protects the host’s genetic material breaks down, making it easily accessible. HIV-1 is a type of retrovirus, called a lentivirus, that can infect non-dividing cells.

HIV-1 delivers its genome into the nucleus by packaging it into a large, cone-shaped structure called a capsid — but the exact mechanism has remained elusive for decades. Travel through the nuclear envelope occurs through, and is regulated by, nuclear pores, doughnut-shaped protein assemblies. Human cells have about 2,000 nuclear pores perforating the nuclear envelope. Some earlier evidence suggested that the capsid remains intact during its delivery into the nucleus — but this created a dimensional conundrum. The cone-shaped HIV-1 capsid is about 120 nanometers long and 60 nm wide — too large, researchers thought, to fit through the opening of the nuclear pore, measured at only 43 nm wide.

Members of the Schwartz Lab at MIT, in the Department of Biology, became interested in this question when a postdoc in the lab used cryo-electron tomography, slicing up sections of frozen cells to examine structures, to show that nuclear pores in the nuclear envelope are larger than 43nm. They deflate and shrink, it turns out, when removed from their native conditions. In native conditions, the nuclear pore complex is about 60nm wide — wide enough to accommodate the HIV-1 capsid.

Knowing that it could fit, a question remained: How can the capsid navigate the dense mesh of spaghetti-like proteins that act like a sieve in the nuclear pore channel? That spaghetti-like mesh allows small cargo to diffuse through, but prevents large cargo from entering unless it is escorted by proteins called nuclear transport receptors.

In an open-access paper published today in Nature, researchers present evidence that the HIV-1 capsid mimics the cell’s transport receptors to traverse the nuclear pore.

To support that conclusion, the researchers showed three things in vitro: that an HIV-1 capsid can deliver cargo through a nuclear pore analog; that the capsid can interact with the sieve of proteins in the nuclear pore channel; and that the capsid targets the nuclear pore in the absence of native transport proteins.

Nuclear transport receptors escort large cargo through the nuclear pore by “batting away” the spaghetti-like mesh of proteins inside the channel — like someone holding your hand and guiding you across a crowded dance floor. The HIV-1 capsid interacts with the spaghetti-like proteins, but its purpose is more like a Trojan horse — the capsid encapsulates the viral cargo, protecting it from detection in the cytoplasm and as it enters the nuclear pore complex.

“What’s really amazing about cells is that they are incredibly complex. What’s really difficult about studying cells is that they are incredibly complex,” jokes co-first author Erika Weiskopf, a graduate student in the Schwartz lab. “Biochemists are constantly trying to find ways to study their system in a simplified context, but still give it a flavor of cell biology.”

To do that, the Schwartz lab collaborated with Dirk Görlich, the director of cellular logistics at the Max Planck Institute for Multidisciplinary Sciences. Görlich is a co-senior author on the paper with MIT’s Boris Magasanik Professor of Biology Thomas Schwartz. Görlich’s lab has produced concentrated droplets of the spaghetti-like proteins found inside the nuclear pore, and those droplets allow and exclude cargo the same way a nuclear pore will. In experiments, fluorescently-labeled cargo did not enter the droplets, but fluorescently-labeled cargo packaged in an HIV-1 capsid was delivered. This indicated that the capsid could deliver cargo through a nuclear pore.

Using a biophysical binding assay, the researchers also showed that the HIV-1 capsid interacts with the proteins inside the channel. Different spaghetti-like proteins are found in different channel sections, such as at the cytoplasmic side’s entrance or only inside the channel; there are 10 such proteins in human cells. The capsid is a promiscuous binder — it can interact with all the spaghetti-like proteins found in the channel.

The capsid can target the nuclear pore complex even without the cell’s transport receptors, indicating that it is not commandeering native transport receptors to find and enter the nuclear pore. The team used a classic assay in the nucleocytoplasmic transport field to collect this evidence: When cells are treated with digitonin, their membranes become porous. Everything in the cytoplasm will leak out of the cells, but the nuclear envelope will remain intact. Despite the absence of native proteins, the capsid was attracted to the nuclear pore complex, a behavior indicative of a nuclear transport receptor.

Although the capsid behaves like a nuclear transport receptor to penetrate the nuclear pore, it is fundamentally different. A transport receptor doesn’t need to conceal material for delivery the way the capsid does to avoid detection.

These findings open new lines of inquiry for what the nuclear pore complex is capable of accommodating.

“The HIV-1 capsid is one of the largest things that we now know can go through the nuclear pore complex intact,” Weiskopf says. “It raises all kinds of questions — what other things could be going through the pore that we thought was impossible?”

Schwartz said another question is whether all of the 2,000 nuclear pores in human cells are identical or whether there is something that makes certain pores more amenable to allowing the capsid through.

The capsid is also known to be unusually elastic, a property that may be key for passage through the pore. Another interesting question for the field is whether the cone-shaped capsid gains entry into the pore by squeezing through.

Although the team has shown that the capsid can enter the pore, what happens at the other end of the channel is still unknown — whether the capsid fully or partially enters the nucleus or breaks down inside the channel. Weiskopf is working on perturbing parts of the capsid or the spaghetti-like proteins to learn more about which interactions are most important for successful capsid entry.

Although these results have expanded our understanding of the nuclear pore, much remains unknown, both for HIV-1 infection and for the transport process through the nuclear pore complex.

“The nuclear pore is such an important element of cell biology, we thought it would be interesting to understand it better — and that’s how we figured out that the pore is much bigger than we anticipated,” Schwartz says. “We will certainly try to see whether we can understand the mechanism of HIV-1 infection, how the capsid is released on the other side of the channel, and what factors are important there — and to what extent you can manipulate it or influence it for therapeutic applications.”