Gene editing of prions done by a virus

Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker disease, fatal insomnia, Kuru disease, and mad cow disease are all neurodegenerative diseases that have a common cause: prions. Although they are rare conditions, a diagnosis guarantees death within a year due to the nature of the disease.

Annually, approximately 500 cases are diagnosed within the U.S. alone, and estimates suggest that the total number of cases worldwide is around 8,000. Worse yet, surveillance of prion disease prevalence and diagnosis varies by country, so there is a possibility that these numbers are underreported. With the severity of the disease, inconsistency of surveillance, and no treatments, addressing prion disease symptoms has become a priority for medical professionals.

The name is an abbreviation of “proteinaceous infectious particle,” referring to prions, which are proteins themselves that can also infect and corrupt other proteins, similar to viruses and bacteria. Due to the infectious nature of prions, a single prion can convert hundreds of normal proteins into prions, thereby exacerbating the disease.

For treatments to be effective, the levels of prions within the brain need to be lowered, and researchers from the University of Toronto’s Tanz Centre for Research in Neurodegenerative Diseases have shown that a virus can be used to help.

The prion protein gene, PRNP, has been the target of therapies aimed at removing prions and alleviating neurodegenerative symptoms. Current treatments to shut off the PRNP gene involve using RNA, the intermediate genetic material that serves as a blueprint for building proteins. Instead of acting as blueprints, special RNA called antisense or silencing RNA are created to act as a genetic counterpart to the PRNP RNA.

When the antisense or silencing RNA binds to the PRNP RNA, the cell recognizes it as a potential threat (since viruses can utilize double-stranded RNA) and destroys the resulting double-stranded RNA. Thus, no prion proteins are produced, resulting in less severe prion disease!

In studies using isolated brain cells, the use of these specialized RNAs was effective in reducing the levels of prion proteins. However, when the RNAs were administered to humans in clinical trials, the RNAs weren’t very effective at entering the brain, their intended target. The blood-brain barrier is famously known for being the most tightly regulated security system in the human body. Very little is allowed to cross the barrier, restricted to vital nutrients for the brain cells and specific immune cells when more help is required during an infection.

Along with that physical hurdle, antisense and silencing RNAs have a short shelf life, as they are less stable than DNA and degrade easily unless protected or maintained. Researchers from the Tanz Centre for Research in Neurodegenerative Diseases at the University of Toronto considered a different approach to stopping the PRNP gene from functioning, using a delivery system to protect the RNA.

The researchers used a virus to transport gene editing technology to remove the PRNP from brain cells. The virus, called a recombinant adeno-associated virus (rAAV), is a single-stranded RNA virus that has its original viral genes replaced with new ones. These modified viruses have become the gold standard in gene therapy due to their ability to naturally target cells and transport genetic information without causing pathogenic side effects. For their hypothesis, the researchers from the Tanz Centre inserted the CRISPR-Cas9 gene system into a rAAV. This gene system uses a guiding RNA and a special enzyme called Cas9.

When the guiding RNA finds its matching DNA sequence in a cell, the Cas9 protein is directed to that location and cuts the DNA-RNA strands. The result is the removal of that DNA section, and the associated gene can no longer be used by the cell.

The CRISPR-Cas9 system installed into the rAAV employed a guiding RNA to target the PRNP gene in brain cells. The goal was to see if using a rAAV designed to remove a dysfunctional gene could be a potential treatment and candidate for clinical trials. As a proof-of-concept study, the team used mice and a Cas9 system equipped with a luminescent tracker to locate the rAAV within the brain.

From their results, they were able to calculate that the CRISPR-Cas9 had an efficiency of approximately twenty percent when cutting out the PRNP gene. Despite the relatively low efficiency, when the researchers checked the protein level of prions, they found a five percent reduction in mice treated compared to those that were not.

The researchers also found that the manner of injecting the rAAV gene therapy mattered. Two injection sites were used: intrathalamic (directly into the brain) and retro-orbital (behind the eye). Surprisingly, when the rAAV was injected retro-orbitally, it was more evenly distributed throughout the brain compared to when it was injected directly into the brain.

To improve the distribution of rAAV in the brain, the team mixed and matched different viral protein shells to determine which one was more effective at entering brain cells. From testing three different lab-made viral shell variants, one, called 9P31, showed the highest distribution, with a 7.5-times increase in distribution compared to the original rAAV used.

These findings are promising but far from a treatment. The team has demonstrated the potential for a rAAV optimized to travel throughout the brain and remove the PRNP gene using gene editing technology.

However, there are limits of efficacy that can be attributed to this work being the first of its kind. Therefore, further studies are needed to optimize the rAAV and gene removal of PRNP and determine if the reduced prion protein correlates with improved disease outcomes.

Despite these limitations, this new technology can impact more than just prion diseases. These rAAV can be studied and modified to address other neurodegenerative diseases that exhibit prion-like mechanisms as well, like Alzheimer’s, Parkinson’s, and Huntington’s disease. This work is just the tip of the iceberg for a new treatment that can lead us closer to a cure for terrible diseases.