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

McDonald’s, Burger King, Wendy’s, Five Guys, Sonic, Shake Shack, In-n-Out. In these fast food restaurants, someone is eating a burger. Unbeknownst to them, that burger came from a cow infected with Bovine Spongiform Encephalopathy, better known as Mmad Ccow Ddisease. Decades later, the same person begins losinges control of their muscles and experiencinges rapid dementia. A year after symptoms first start, they pass away, a victim of prion disease. Prion diseases are a collection of neurodegenerative diseases caused by misfolded proteins known as prions. Normally-folded prion proteins (PrP) are abundant in the brain and are thought to have no function. 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 Bovine Spongiform Encephalopathy (Mad Cow Disease) in cattle, which are known to be transmissibletransmitable to humans [1].

 A unique feature of prion proteins is that their misfolded variant can cause neighboringother prion proteins to misfold, causing a chain reaction that kills off neurons and creates openings and pockets in the brain that gives it a “spongy” appearance [2]. Misfolded prions can be acquired through genetics affecting the stability of PrP, spontaneous misfolding, or ingestion from contaminated meat or surgical tools. Prion diseases are always fatal since prions cannot be killed by the immune system, nor by heat, radiation, or chemotherapy, as they are not alive and are incredibly stable [3]. 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 the disease [4, 5]. Thus, they sought to find ways to limit PrP expression and/or get rid of Prnp, the gene that codes for PrP. 

A study in 2019 by Raymond et al. studied the use of antisense oligonucleotides (ASOs) as a treatment [6]. ASOs are short, engineered strands of RNA that can bind to a specific mRNA and decrease protein expression. They are a very common treatment when it comes to monogenic disorders [7]. In this study, the researchers tested single and multiple injections of ASOs into the cerebrospinal fluid of prion-infected mice. They injected a group right at infection and another group 120 days after infection. The researchers discovered that ASO treatment does decrease prion disease development and extended survival 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 [6]. 

Another study in 2025 by An et al. decided to take a different approach to limit PrP expression. Instead of using CRISPR, a popular gene editing tool, they modified it to be able to swap out single nucleotide bases instead of cutting and inserting large sections of DNA, calling them cytosine base editors (CBEs). They chose not to use CRISPR because 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 [8]. CRISPR also has the capability to accidentally edit the wrong gene, potentially causing untold effects [9]. People can have a negative immune response in response to the Cas9 protein used in CRISPR which is derived from bacteria as well[10, 11]. This made it imperative to develop a smaller, more precise, and more accurate gene editing tool like CBEs. CBEs swap out a single base from cytosine-guanine for adenine-thymine to change an amino acid codon into a stop codon. They chose to edit amino acid 37 in Prnp because the variant (R37X) had been previously found in humans and most likely was not harmful. To ensure this technology worked before testing with living animals, the authors edited samples of human cells and validated that it did in fact perform as intended. They transported the CBE through a kind of AAV called AAV-PHP.eB, which could pass through the blood-brain barrier undetected. The scientists injected the virus retro-orbitally into mice one week before infecting them with human misfolded prions. Treated mice survived 44-59% longer than untreated mice. Overall, after 100 days, 20% of the Prnp had the Prnp R37X edit and there was a 31% decrease in PrP [8]. 

Neumann et al. (2024) instead 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 AAV-PHP.eBs as transport, CHARM is able to locate Prnp, recruit the methyltransferases to methylate the gene, and then shut itself off. They injected CHARM retro-orbitally into mice to discover the efficacy of the gene silencer. After 6-13 weeks, they found that 70-90% of Prnp was methylated and PrP levels decreased by 60-80% [12]. 

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 like astrocytes and it is simply inferred.  Unfortunately for both CHARM and CBEs, AAVs have been known to be deadly at high doses with deaths ranging 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 1 × 1013 vg/kg, there is still potential long term risk towards using AAVs [8, 12, 13]. CHARM also did not test using infected mice, and CBEs only were injected before infecting the mice, not accounting for any potential variables post-infection. As for ASOs, although they last decently long, they don’t last forever. They also cannot cross the blood brain barrier, which means they must be injected intrathecally or through the spinal cord into the cerebrospinal fluid. This means that the patient will have to get repeatedly injected through the spine, which tends to be invasive, impractical, and expensive [6].  

Overall, none of these therapies have been used on humans to treat prion disease, showing that 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, increasing the risk of transmitting prion disease through surgical tools especially since prions are not killed through standard sterilization practices. Furthermore, there is debate of the function of prion proteins, and some argue that the lack thereof could have detrimental consequences [14, 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. 

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