Today, biologist Sonia Vallabh is an influential figure in the field of prion research. She and her husband, Eric Minikel, founded the Prion Alliance at the Broad Institute of MIT and Harvard to spearhead efforts to develop therapies for prion disease.
Neither Vallabh nor Minikel began their careers planning to study prion disease or even planning to work in biology. In 2010, the couple were recently married. Vallabh was in her second year of law school at Harvard University and Minikel was enrolled in a Master’s degree program in city planning and transportation at the Massachusetts Institute of Technology (MIT). Then Vallabh’s mother developed a mysterious illness.
“It was not immediately clear that she had a terminal illness,” Vallabh recalled. “We spent the whole year of 2010 rushing around, trying to figure out what was happening because the rapid decline was really evident, but the cause was still unclear.”
From mild symptoms in January, Vallabh’s mother’s condition quickly worsened, and she passed away before the end of the year. She was 52 years old.
The following year, Vallabh learned what had caused her mother’s illness: genetic prion disease. Vallabh immediately decided to undergo genetic testing, and by the end of 2011, she received life-altering results. Vallabh also carries the mutation in the human prion protein gene that puts her at extremely high risk for developing this fatal disease.
“It wasn’t very long after that that things started to shift for us. Initially, we just wanted to learn more, just wanted to understand what was known about prion disease, and wanted to engage more in the research side of things,” said Vallabh. “The clinical picture was then — and still is today — just incredibly bleak. But the research arena had a significantly different tone and a different energy.”
While clinicians had few answers about prion disease, Vallabh learned that research groups around the world were studying the disease’s biochemistry and pathogenesis, and efforts to develop diagnostics and therapeutics were underway in animal models.
Vallabh and Minikel enrolled in night classes, and before long, they quit their respective jobs and took entry level positions in biomedical research. Initially, they simply wanted to understand prion disease, but their interests shifted towards finding a treatment. In 2014, the pair began PhD programs at the Broad Institute, and once they graduated, they established a joint lab at the institute to study the mechanisms of prion disease and search for biomarkers. Above all else, they want to develop an effective therapy.
As a patient-scientist, Vallabh brings a unique perspective to prion disease research. While research has historically focused on treatments for symptomatic disease, Vallabh has a special interest in also developing preventative treatments.
“As we learn more about prion disease in our own lab and in our own hands, what we've seen is that early intervention really matters,” said Vallabh. “It makes sense that when we're talking about something as complex as the human brain, you really do want to preserve, as best you can, what you don't understand and can never rebuild.”
Indeed, by the time symptoms appear, a diagnosis is made, and treatment starts, substantial damage has already occurred. Prion diseases progress with astonishing swiftness; patients rarely survive for more than a year after symptom onset (1).
Investigating preventative measures requires leveraging genetic information to show who's at risk ahead symptom onset. “It also means a certain way of thinking about drug development, where we think about creative ways to test drugs in healthy people for their ability to prevent disease,” said Vallabh.
Choosing a target
The terminology surrounding prions is somewhat complicated. All humans have a gene (PRNP) that codes for the normal prion protein, also known as cellular prion protein, or PrPC. This cell surface protein is abundant in the brain and is not harmful. Sometimes, however, the normal prion protein becomes misfolded; this form is called a prion, or PrPSc. The misfolding can be spontaneous or due to genetic mutations.
In extremely rare cases, prion disease can be acquired, as in some instances in which these misfolded proteins were accidentally introduced into patients during medical procedures (2). The misfolded form of the normal protein is pathogenic, inducing normal prion proteins in the brain to misfold as well in a process known as prion propagation. Prions can be misfolded in different ways, resulting in various three-dimensional structures, and prion disease subtypes have slightly different symptoms. However, all human prion diseases result from the misfolding of the normal prion protein, and all result in neurodegeneration and eventual death.
Research groups are exploring a variety of approaches to target this protein and its downstream effects on the brain; Vallabh and Minikel plan to design a therapy to reduce the amount of prion protein in the brain. There are strong proof-of-concept studies to support this as a promising strategy, said Vallabh.
“Prion protein knockout mice have existed for decades,” said Vallabh, “If you try to infect those mice with prions the way you would infect a normal wild type mouse, they don't get sick. The substrate is sort of like fuel for a fire. If you don’t have that substrate, you can't propagate prions, and you can't get sick.”
Strikingly, mice that lack this protein develop and reproduce normally, without any obvious impairments (3).
The substrate is sort of like fuel for a fire. If you don’t have that substrate, you can't propagate prions, and you can't get sick.
- Sonia Vallabh, Broad Institute
Since the normal prion protein exists in healthy people, Vallabh said that it’s an attractive target for developing preventative therapies. She and her team can test whether a treatment lowers prion protein levels in people at risk for the disease without waiting for them to get sick first.
Even though scientists knew exactly which protein they wanted to target, figuring out the best way to reduce prion protein levels in a human brain is no easy task. In 2014, Vallabh and Minikel began speaking with Ionis Pharmaceuticals about a potential therapeutic strategy using antisense oligonucleotides (ASO) to reduce target RNA levels in the brain. Ionis’ ASO bind to the disease-causing RNA and recruit an enzyme that destroys the RNA, preventing it from being translated into a disease-causing protein (4). Researchers at Ionis and other institutions were working on problems with toxicity and drug delivery that had plagued early research on ASO.
Vallabh and Minikel established a collaboration with the company in 2016. In 2019, the research team produced an ASO that ameliorates disease caused by the Rocky Mountain Laboratory strain of prions in mice (5). For these mice, a single dose of the ASO extended survival by 55 percent when given before the onset of symptoms.
Further work on ASO demonstrated that these therapeutics also increased survival of mice infected with four other prion strains. They even had some benefit when given after symptom onset (4).
According to Vallabh, the efficacy of the ASO against several different strains is especially promising and is not necessarily true of many potential therapeutics. “Small molecule drug development has run into so many challenges that really spring from the structural mysteries around misfolded prions and the fact that they come in different three-dimensional conformations,” she said. “Probably every case of prion disease contains a cloud of different conformations. And these small molecules, through we know not what mechanism, seem to selectively target some conformations. So, you can get a rebound of the others and basically end up with the disease evolving rather than being stopped in its tracks.”
Now that researchers have established that ASO can lower prion protein RNA in mice, Vallabh said that the question now is one of therapeutic window: Can researchers develop an ASO that can reduce prion protein enough, in a large enough area of the human brain, while still being tolerated?
Although there is still work to be done, Vallabh anticipates that, “the first-in-human dosing is probably not that far away.”
More than one way to fight a prion
John Collinge, a neurologist at University College London, has been intrigued by prions for more than 30 years. He started off studying the genetics of familial forms of prion disease and soon afterwards began to explore potential therapeutics for different forms of prion disease.
Instead of trying to reduce the amount of normal prion protein in the brain like Vallabh and Minikel, Collinge’s team took a different approach. “Our strategy was to restrict the flow of the normal prion protein into the chain reaction that produces the prions and produces neurotoxicity,” he said.
The team needed to figure out how to prevent the conversion of the normal prion protein into a harmful, misfolded form. “We know that the normal protein has to basically completely unfold and refold into the new form to form prions. So anything that binds to the normal form of the protein should reduce chances of that happening.”
Creating molecules that stick to the normal prion protein was no easy task, however. “We tried very hard over a number of years, actually, to develop small molecule drugs, but we weren't successful because the prion protein doesn't really have traditional drug binding sites,” said Collinge. “What we were doing in parallel, and that's turned out to be more successful, was to use a monoclonal antibody. Obviously, antibodies are molecules that have evolved to bind to proteins rather efficiently.”
The normal protein has to basically completely unfold and refold into the new form to form prions.
- John Collinge, University College London
In 2003, Collinge’s team demonstrated an early success: antiprion protein antibodies substantially delayed the onset of disease in prion-infected mice (6). While untreated mice succumbed to the disease after about 200 days, mice that received the antibody treatment twice a week were still healthy 500 days after infection.
In October 2018, the team initiated a first-in-human trial (7). Six patients with Creutzfeldt–Jakob prion disease received intravenous infusions of the new antibody therapy, which is called PRN100.
Since only six patients were treated, it was difficult to determine whether or not the treatment altered the course of the disease. Although all patients’ health generally declined over the course of treatment, and there was not a statistically significant difference between treated patients and historical cases, which stood in for the untreated control group, “the rate of decline did seem to slow when they were in the therapeutic range we were aiming for,” Collinge said.
PRN100 was tolerated well and reached the target concentration in the cerebrospinal fluid, indicating that it could cross the blood-brain barrier. Overall, Collinge thinks that PRN100 warrants further study; he and his team want to do a Phase 2 trial with 50 patients in order to more fully explore the drug’s efficacy. Collinge said that for future trials, catching patients early in the course of disease will be critical.
“When patients have gotten to a fairly advanced stage in the neurodegeneration, it may be difficult, if not impossible, to stop, even if you stop prions propagating,” he said. “There's a lot of cell death occurring at that point in time. And as brain cells die, they release all sorts of toxic substances that will cause more cells around them to die, and then those cells die, and they produce even more toxins, so you then have an irreversible process.”
Old drugs with new tricks
Sabine Gilch, an infection biologist at the University of Calgary, is studying the interactions between prions and cellular cholesterol metabolism with an eye towards finding new therapies.
Her studies build on early work demonstrating that cellular cholesterol was related to prion formation (8). During graduate school at Technical University of Munich, Gilch showed that inhibiting cholesterol recycling in cells reduced prion propagation (9). She also showed that prion-infected neurons ramp up cholesterol synthesis gene expression (10).
Even with the target in mind, however, it has not been straightforward to translate these findings into animal versions of the disease. Drugs to reduce cholesterol synthesis or extract cholesterol from cell membranes were effective in cells, but didn’t seem to have any benefits for prion-infected mice.
Gilch wasn’t ready to give up on cholesterol modulation yet, though. She kept searching for new targets that could regulate the amount of cholesterol in cells and eventually came across cholesterol 24 hydroxylase, or Cyp46A1. This enzyme converts cholesterol into a form that can cross membranes and exit the brain.
Further experiments showed that the amount of Cyp46A1 was reduced in prion-infected cell cultures and in the brains of prion-infected mice, at least at the later stages of disease (11). Gilch also found reduced amounts of Cyp46A1 protein in postmortem brain tissues from patients with Creutzfeldt–Jakob disease compared to postmortem tissues from people without the disease.
In order further investigate the role of Cyp46A1, Gilch repurposed a drug called efavirenz. This drug has been FDA approved to treat HIV for decades, but more recently was shown to activate Cyp46A1 as well. Gilch found that this drug reduced prion propagation in vitro and modestly extended the lifespans of prion-infected mice (11).
This study used a mouse-specific prion strain, but Gilch said that work is in progress to determine if efavirenz is also beneficial for other strains of prions, including those that affect humans. She wants to continue to explore the exact nature of the relationship between cholesterol and prion propagation.
A more familiar adversary
While most researchers are focusing on the prions themselves, Julie Moreno, a molecular biologist at Colorado State University, thinks that targeting downstream effects presents another promising therapeutic approach. Her target is a familiar one in the field of neurodegenerative disease: inflammation. In the case of prion disease, the accumulation of prions leads to neuroinflammation, which seems to play a role in neuronal death (12,13).
During graduate school, Moreno studied the relationship between environmental toxicants and Parkinson’s disease. “I was really interested in the brain, and how cells respond to certain stressors and cause disease pathology,” she said.
She began studying prions after taking a position at the University of Cambridge. “My advisor, Giovanna Mallucci, sold me on this idea that studying prion disease is one of the best ways for us to not only improve our understanding of prion disease, but also other neurodegenerative diseases.”
Although rodent models of neurodegenerative diseases have come to more closely represent the diseases over the years, they still can’t fully recapitulate them. For instance, researchers might use a specific gene mutation to mimic some features of Alzheimer’s disease in mice, but in humans, most cases of Alzheimer’s disease are not caused by a single gene mutation; the processes driving the disease will not match exactly.
“Mice that are exposed to prions actually get the disease. They don’t have to be genetically modified. If you give them a little seed of prions, they will replicate it and propagate it in the brain, just like a human would,” Moreno said. “When you think of other diseases of the brain, like Alzheimer's or Parkinson's, there's not as good of a model. Prion disease isn't even really a model — people in the field sometimes get mad if you call it that — because the mice actually get the disease.”
While prion propagation is the main hallmark of prion disease, neuroinflammation is also an important feature (14–16). Moreno teamed up with the company Sachi Bioworks to test whether Nanoligomers — peptide molecules designed to downregulate expression of specific genes — targeting the proinflammatory NF-κB transcription factor and NLRP3 inflammasome protein could be useful in prion disease. In a recent preprint, she reported that these drugs seemed to improve neuronal survival and cognitive abilities in the later stages of the disease, although lifespan increased only modestly (17).
Since the Nanoligomers seemed to have a good safety profile, Moreno plans to explore whether higher doses produce a stronger effect.
Moreno acknowledges that this isn’t a silver bullet. To stop the disease completely will likely require stopping the accumulation of the toxic prions. Until scientists figure out how to do that, however, reducing neuroinflammation might ease symptoms and improve quality of life. Therapies that address neuroinflammation may need to be combined with other therapies to produce the best outcomes for patients.
“This isn’t going to just be one hit,” she said. “You can't just target one thing. You need to target multiple signaling pathways to stop the toxicity.”
For now, prion disease remains a devastating diagnosis. One day, however, thanks to the dedication of prion disease researchers, new therapies may enable Vallabh and others like her to live long and healthy lives.
References
- Tejedor-Romero, L. et al. Survival Patterns of Human Prion Diseases in Spain, 1998–2018: Clinical Phenotypes and Etiological Clues. Frontiers in Neuroscience 15, (2022).
- UCL. Acquired prion disease. National Prion Clinic (2021). at <https://www.ucl.ac.uk/national-prion-clinic/acquired-prion-disease>
- Büeler, H. et al. Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 356, 577–582 (1992).
- Liang, X.-H., Sun, H., Nichols, J. G. & Crooke, S. T. RNase H1-Dependent Antisense Oligonucleotides Are Robustly Active in Directing RNA Cleavage in Both the Cytoplasm and the Nucleus. Mol Ther 25, 2075–2092 (2017).
- Raymond, G. J. et al. Antisense oligonucleotides extend survival of prion-infected mice. JCI Insight 4, e131175
- White, A. R. et al. Monoclonal antibodies inhibit prion replication and delay the development of prion disease. Nature 422, 80–83 (2003).
- Mead, S. et al. Prion protein monoclonal antibody (PRN100) therapy for Creutzfeldt–Jakob disease: evaluation of a first-in-human treatment programme. The Lancet Neurology 21, 342–354 (2022).
- Taraboulos, A. et al. Cholesterol depletion and modification of COOH-terminal targeting sequence of the prion protein inhibit formation of the scrapie isoform. J Cell Biol 129, 121–132 (1995).
- Gilch, S., Bach, C., Lutzny, G., Vorberg, I. & Schätzl, H. M. Inhibition of cholesterol recycling impairs cellular PrPSc propagation. Cell Mol Life Sci 66, 3979–3991 (2009).
- Bach, C. et al. Prion-induced Activation of Cholesterogenic Gene Expression by Srebp2 in Neuronal Cells *. Journal of Biological Chemistry 284, 31260–31269 (2009).
- Ali, T. et al. Oral administration of repurposed drug targeting Cyp46A1 increases survival times of prion infected mice. Acta Neuropathologica Communications 9, 58 (2021).
- Crespo, I., Roomp, K., Jurkowski, W., Kitano, H. & del Sol, A. Gene regulatory network analysis supports inflammation as a key neurodegeneration process in prion disease. BMC Syst Biol 6, 132 (2012).
- Li, B., Chen, M. & Zhu, C. Neuroinflammation in Prion Disease. Int J Mol Sci 22, 2196 (2021).
- Inhibition of neuroinflammatory nitric oxide signaling suppresses glycation and prevents neuronal dysfunction in mouse prion disease. doi:10.1073/pnas.2009579118
- Carroll, J. A., Striebel, J. F., Race, B., Phillips, K. & Chesebro, B. Prion Infection of Mouse Brain Reveals Multiple New Upregulated Genes Involved in Neuroinflammation or Signal Transduction. Journal of Virology 89, 2388–2404 (2015).
- Monzón, M., Hernández, R. S., Garcés, M., Sarasa, R. & Badiola, J. J. Glial alterations in human prion diseases. Medicine (Baltimore) 97, e0320 (2018).
- Risen, S. J. et al. Targeting neuroinflammatory pathways using NanoligomersTM is neuroprotective in prion disease. 2022.09.26.509513 Preprint at https://doi.org/10.1101/2022.09.26.509513 (2022)