Works Like A CHARM: A Step Towards Finding a Cure for Deadly Prion Diseases

Hong-Duc Pho

Thomas Jefferson High School for Science and Technology

McDonald’s, Burger King, Wendy’s, Five Guys. All these restaurants have one thing in common: burgers. Everyday, people buy and eat these burgers across America. Unbeknownst to them, some of those burgers came from a cow infected with a kind of prion disease called Bovine Spongiform Encephalopathy, better known as Mad Cow Disease. Decades later, the same person begins losing control of their muscles and experiencing dementia. A year after symptoms first start, they pass away—another victim of prion disease. 

Prion diseases are a collection of neurodegenerative diseases caused by misfolded proteins in the brain known as prions. Classic Creutzfeldt-Jakob disease, variant Creutzfeld-Jakob disease, Gerstmann-Straussler-Scheinker disease, Kuru, and Fatal Familial Insomnia are all prion diseases known to affect humans, characterized by dementia and nervous system dysfunction, with patients dying within a year. There are also records of prion diseases within animals, such as scrapie in goats and sheep and Mad Cow Disease in cattle, which are known to be transmissible to humans [11].

 Regular prion proteins (PrP), the normally folded version of prions, are abundant in the brain and are thought to have no function. A unique feature of PrP is that their misfolded variant can cause other PrP to misfold, causing a chain reaction that kills off neurons and creates pockets in the brain that gives it a “spongy” appearance [7]. It’s this chain reaction and the formation of pockets that causes prion disease and all of its symptoms, including rapid dementia. Misfolded PrP can be acquired through ingestion from contaminated meat, surgical tools, spontaneous misfolding, or genetics affecting the stability of PrP. Prion diseases are always fatal since prions cannot be killed by the immune system, nor by heat, radiation, or chemotherapy. This is because prions are not alive and are incredibly stable [14]. Scientists, however, found that mice lacking PrP are insusceptible to prion diseases and that decreasing PrP levels in the brain can both halt and counter prion diseases [2, 9]. Thus, researchers sought to find ways to limit PrP expression by getting rid of Prnp, the gene that codes for PrP. 

When it comes to gene editing, CRISPR is the standard, but in this case, it has its limitations. For one, it is too large to be transported into the brain via an adeno-associated virus (AAV), which is a small, non-pathogenic virus that acts as a way to transport genetic material into the body [1]. CRISPR can also accidentally edit the wrong gene, potentially causing untold effects [6]. Additionally, people can have a negative immune response in response to the Cas9 protein used in CRISPR, which is derived from bacteria [4, 13]. This made it imperative for scientists to find an alternative solution for deactivating Prnp. 

A 2024 study by Neumann et al. formulated an epigenetic editing tool called CHARM (Coupled Histone tail for Autoinhibition Release of Methyltransferase). This technology takes advantage of the body’s ability to silence genes or turn them “off” using enzymes called DNA methyltransferases. Using AAVs as transport, CHARM is able to locate Prnp, recruit the methyltransferases to turn off the gene, and then shut itself off. They injected CHARM into mice to discover the efficacy of the gene silencer. After 6-13 weeks, they found that 70-90% of Prnp was turned off and PrP levels decreased by 60-80% [10]. 

Additionally, Raymond et al. (2019) studied the use of antisense oligonucleotides (ASOs) as a treatment for prion disease [12]. ASOs are short, engineered strands of RNA that can stop PrP from being synthesized by binding to its mRNA. They are a very common treatment for monogenic disorders (inherited conditions caused by a single gene/protein) [8]. In this study, the researchers tested single and multiple injections of ASOs into the spinal fluid of prion-infected mice. They injected a group at the onset of infection and another group 120 days after infection. The researchers discovered that ASO treatment decreases prion disease development and extends survival time by around 55-98%. Lastly, they found that large doses have similar effects in extending mice survival, supporting the idea that ASOs can remain in the brain for extended periods of time [12]. 

However, a 2025 study by An et al. decided to take a different approach to limit PrP expression. Instead of using CRISPR, they modified it to be able to swap out single nucleotide bases instead of cutting and inserting large sections of DNA, calling it a cytosine base editor (CBE). CBEs swap a cytosine-guanine base pair for an adenine-thymine base pair to change an amino acid codon into a stop codon. To ensure this technology worked before testing with living animals, the authors edited samples of human cells and validated that it performed as intended. They transported the CBE through a special kind of AAV which could pass through the blood-brain barrier, the brain’s security system which screens for foreign bodies, undetected. The scientists injected the virus into mice one week before infecting them with prions from humans. Treated mice survived 44-59% longer than untreated mice. Overall, after 100 days, 20% of the Prnp was edited and there was a 31% decrease in PrP [1]. 

All of these studies have unique approaches towards lowering PrP, each with varying levels of success and caveats. For instance, even though CHARM has the most success, it doesn’t look into Prnp silencing in brain cells and it is simply inferred. Furthermore, the AAVs used to transport CHARM and CBEs have been known to be deadly at high doses with deaths occurring from days to weeks due to heart, lung, and liver failure. Even though those deaths are from doses around 1.1 × 1014vg/kg (vector genomes per kilogram of body weight) and CHARM and CBEs were injected with only around a tenth of that amount, there is still potential long term risk towards using AAVs [1, 10, 5]. Neumann et al. also did not test CHARM using infected mice, and CBEs were only injected before infecting the mice, which did not account for any potential variables post-infection. As for ASOs, although they can last for long periods of time inside the brain, they don’t last forever. They also cannot cross the blood brain barrier, which means they must be injected through the spinal cord into the spinal fluid. This means that the patient will have to get repeatedly injected through the spine, which tends to be invasive, impractical, and expensive [1].  

Overall, none of these therapies have been used on humans to treat prion disease, showing that further experimentation still needs to be done before a successful and safe cure is formulated. Despite the fact that prion disease is rare, brain surgeries, including Elon Musk’s Neuralink, are expected to become more and more common. This could increase the risk of transmitting prion disease through surgical tools, especially since prions are not killed through standard sterilization practices. Furthermore, there is debate around the function of PrP, and some argue that the lack thereof could have contributed to some of the symptoms of prion disease  [3, 15]. Fortunately, these breakthroughs in epigenetics aren’t just a step towards curing prion diseases, but a step towards curing a variety of monogenic disorders and similar neurodegenerative diseases. 

References

[1] An, M., Davis, J. R., Levy, J. M., Serack, F. E., Harvey, J. W., Brauer, P. P., Pirtle, C. P., Berrios, K. N., Newby, G. A., Yeh, W.-H., Kamath, N., Mortberg, M., Lian, Y., Howard, M., DeSouza-Lenz, K., Guzman, K., Thai, A., Graffam, S., Laversenne, V., . . . Liu, D. R. (2025). In vivo base editing extends lifespan of a humanized mouse model of prion disease. Nature Medicine, 31, 1319-1328. https://doi.org/10.1038/s41591-024-03466-w

[2] Büeler, H., Aguzzi, A., Sailer, A., Greiner, R.-A., Autenried, P., Aguet, M., & Weissmann, C. (1993). Mice devoid of prp are resistant to scrapie. Cell, 73(7), 1339-1347. https://doi.org/10.1016/0092-8674(93)90360-3

[3] Collinge, J., Whittington, M. A., Sidle, K. C. L., Smith, C. J., Palmer, M. S., Clarke, A. R., & Jefferys, J. G. R. (1994). Prion protein is necessary for normal synaptic function. Nature, 370(6487), 295-297. https://doi.org/10.1038/370295a0

[4] Crudele, J. M., & Chamberlain, J. S. (2018). Cas9 immunity creates challenges for CRISPR gene editing therapies. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-05843-9

[5] Duan, D. (2023). Lethal immunotoxicity in high-dose systemic AAV therapy. Molecular Therapy, 31(11), 3123-3126. https://doi.org/10.1016/j.ymthe.2023.10.015

[6] Guo, C., Ma, X., Gao, F., & Guo, Y. (2023). Off-target effects in crispr/cas9 gene editing. Frontiers in Bioengineering and Biotechnology, 11. https://doi.org/10.3389/fbioe.2023.1143157

[7] Hughes, D., & Halliday, M. (2017). What is our current understanding of prpsc-associated neurotoxicity and its molecular underpinnings? Pathogens, 6(4), 63. https://doi.org/10.3390/pathogens6040063

[8] Lauffer, M. C., Van Roon-Mom, W., & Aartsma-Rus, A. (2024). Possibilities and limitations of antisense oligonucleotide therapies for the treatment of monogenic disorders. Communications Medicine, 4(1). https://doi.org/10.1038/s43856-023-00419-1

[9] Minikel, E. V., Zhao, H. T., Le, J., O'Moore, J., Pitstick, R., Graffam, S., Carlson, G. A., Kavanaugh, M. P., Kriz, J., Kim, J., Ma, J., Wille, H., Aiken, J., McKenzie, D., Doh-ura, K., Beck, M., O'Keefe, R., Stathopoulos, J., Caron, T., . . . Vallabh, S. M. (2020). Prion protein lowering is a disease-modifying therapy across prion disease stages, strains and endpoints. Nucleic Acids Research, 48(19), 10615-10631. https://doi.org/10.1093/nar/gkaa616

[10] Neumann, E. N., Bertozzi, T. M., Wu, E., Serack, F., Harvey, J. W., Brauer, P. P., Pirtle, C. P., Coffey, A., Howard, M., Kamath, N., Lenz, K., Guzman, K., Raymond, M. H., Khalil, A. S., Deverman, B. E., Minikel, E. V., Vallabh, S. M., & Weissman, J. S. (2024). Brainwide silencing of prion protein by aav-mediated delivery of an engineered compact epigenetic editor. Science, 384(6703). https://doi.org/10.1126/science.ado7082

[11] Prusiner, S. B. (1998). Prions. Proceedings of the National Academy of Sciences, 95(23), 13363-13383. https://doi.org/10.1073/pnas.95.23.13363

[12] Raymond, G. J., Zhao, H. T., Race, B., Raymond, L. D., Williams, K., Swayze, E. E., Graffam, S., Le, J., Caron, T., Stathopoulos, J., O'Keefe, R., Lubke, L. L., Reidenbach, A. G., Kraus, A., Schreiber, S. L., Mazur, C., Cabin, D. E., Carroll, J. B., Minikel, E. V., . . . Vallabh, S. M. (2019). Antisense oligonucleotides extend survival of prion-infected mice. JCI Insight, 4(16). https://doi.org/10.1172/jci.insight.131175

[13] Wagner, D. L., Amini, L., Wendering, D. J., Burkhardt, L.-M., Akyüz, L., Reinke, P., Volk, H.-D., & Schmueck-Henneresse, M. (2018). High prevalence of streptococcus pyogenes cas9-reactive T cells within the adult human population. Nature Medicine, 25(2), 242-248. https://doi.org/10.1038/s41591-018-0204-6

[14] Will, R. G., & Ironside, J. W. (2016). Sporadic and infectious human prion diseases. Cold Spring Harbor Perspectives in Medicine, 7(1), a024364. https://doi.org/10.1101/cshperspect.a024364

[15] Wulf, M.-A., Senatore, A., & Aguzzi, A. (2017). The biological function of the cellular prion protein: An update. BMC Biology, 15(1). https://doi.org/10.1186/s12915-017-0375-5