Synopsis: Shortly after Sonia Vallabh and Eric Minikel were newly married in 2009, Vallabh's mother died of hereditary prion disease, and Vallabh was also found to carry the disease-causing gene. In order to save themselves and save more patients, the two men with no biomedical background quit their jobs and started from scratch. They continue to make progress in their research, and now they are working with their team to develop an epigenetic editing tool, CHARM, that silences the gene encoding prion proteins in the brain, providing a potential therapeutic strategy for prion diseases and other neurodegenerative diseases.
In 2009, Sonia Vallabh, who was pursuing a professional diploma in law at Harvard Law School, had a happy moment in her life when she married Eric Minikel, who is pursuing a master's degree in urban planning and transportation at the Massachusetts Institute of Technology. But just a year later, a major event changed the couple's lives forever.
▲Eric Minikel(左)与Sonia Vallabh(右)(图片来源:博德研究所)
Fatal misfolding
Vallabh's mother fell ill. At first, her vision was blurred, but soon symptoms took hold, and Vallabh's mother's health deteriorated dramatically, and she lost the ability to walk, communicate with people, and eat ...... Just 10 months after the onset of symptoms, Vallabh's mother died in fear. But during this time, no one knew what the true cause was, until the autopsy report told Vallabh an extremely brutal result.
她的母亲死于致死性家族性失眠症（fatal familial insomnia）——一种遗传性朊病毒病。
In our brains, there is a class of cell surface proteins called prion proteins (PrP). Under normal circumstances, PrP is not a bad character and does not have health consequences. However, as soon as PrP changed its conformation, the "dominoes" began to fall. Misfolded PrP forms toxic aggregates that then spread throughout the brain, leading to neuronal death, a deadly and incurable disease known as prion disease.
The prion disease we are most familiar with is the contagious mad cow disease; In addition, other prion diseases can also be caused by genetic mutations, such as the fatal familial insomnia of Vallabh's mother, which is the result of a mutation in the PRNP D178N gene. The PRNP D178N mutation is autosomal dominant, meaning that Vallabh has a 50% chance of inheriting this lethal gene.
Do you keep such worries in your heart, or do you take the initiative to understand your fate? A year later, Vallabh chose to toss the fateful coin. Genetic testing revealed that Vallabh also carried the PRNP D178N mutation. In other words, Vallabh is saddled with a "time bomb" that can "detonate" at any stage of his life, and no one can predict the time of the onset of the disease, but once the symptoms appear, the disease will progress extremely quickly, and what awaits Vallabh at that time can only be death.
Start from scratch and change your life against the sky
Faced with such a "checkmate" situation, the most logical thing for Vallabh, who has no biomedical background, is to continue his work and life while looking forward to a therapeutic breakthrough in the field of prions - after all, although it is not a hot field, there are still some scientists around the world who are studying prion diseases. If luck is right, Vallabh has decades to wait.
But in a race against time, Vallabh and her husband, Minikel, have chosen a path to try to change their lives – researching prion diseases on their own, looking for a way to illuminate themselves and save more patients.
In 2012, Vallabh and Minikel quit their jobs to study biology from scratch and landed their first basic research job at Massachusetts General Hospital. They soon realized that more scientific knowledge was needed to stand a chance of success. In 2014, both applied for a PhD at the prestigious Broad Institute, where they graduated five years later and set up their own laboratory at the Broad Institute to begin research on rare prion diseases.
Unlike some scientists, who use the funding cycle as a research time limit, their deadline is a countdown to the "bomb" that kicks in at any time in Vallabh's body. Amazingly, the halfway scientists made progress in their research.
In 2019, the couple and collaborators proposed a new strategy for monitoring PrP levels in cerebrospinal fluid using existing tools in the journal PNAS; A year later, in the journal Nucleic Acids Research, they proposed that antisense oligonucleotides (ASOs)-mediated PrP inhibition of less than 25% could prolong the survival and delay the onset of disease in prion-infected mice. It is worth mentioning that this ASO-based drug candidate, ION717, is currently in clinical trials.
At the same time, Vallabh and Minikel didn't stop exploring. In collaboration with Professor Jonathan Weissman of the Whitehead Institute of Biomedical Research, another research breakthrough for the couple is featured in Science today! The research team collaborated to develop an epigenetic editing tool called CHARM, which can silence genes encoding PrP throughout the brain, providing a potential therapeutic strategy for patients with deadly prion diseases and other neurodegenerative diseases caused by the aggregation of harmful proteins.
New Epigenetic Editor
▲Schematic diagram of the newly researched CHARM epigenetic editor (Image source: Reference [1])
The starting point of this research can be traced back to CRISPRoff, a gene silencing tool published by Weissman in the journal Cell in 2021. One way to inhibit PrP production is to silence the gene of interest. CRISPRoff, on the other hand, uses the building blocks of CRISPR gene editing technology and uses Cas9 nuclease to cleavage the target DNA, directing the editor to the target gene. CRISPRoff contains a DNA methyltransferase domain (DNMT3A) that enables long-term silencing of target genes through epigenetic modifications of site-directed methylation.
Previous studies have confirmed that CRISPRoff can stably and effectively silence genes encoding PrP. However, several drawbacks of CRISPRoff limit its application.
The first is the issue of size. We know that adeno-associated viruses (AAVs) are common carriers for drug delivery to the brain, and like any "freighter," they are limited in the size of the "cargo" they can carry. Unfortunately, Cas9 protein is a large cargo and cannot be delivered with a single AAV vector. Therefore, the compromise is to increase the dose and load with multiple AAV vectors, which in turn leads to increased cytotoxicity. Another problem is that long-term expression of the CRISPRoff editor carries the risk of harmful immune responses and off-target effects.
Therefore, Weissman's goal with Vallabh and Minikel was to create a new editing tool that was not only equally effective, but also small enough and safe enough to minimize errors in gene silencing.
The first thing to solve is the size problem. The Cas9 used by CRISPRoff was so large that the team's solution was to abandon CRISPR and instead create an epigenomic editor called ZFPoff, which is based on zinc finger protein (ZFP). Among them, the zinc finger protein plays the role of Cas9, guiding the editing tool to reach the target gene. Due to its much smaller size, a single AAV can be used to deliver new editors. At the same time, because zinc finger proteins are commonly found in human cells, there is no need to worry about the risk of autoimmune reactions.
Once the delivery problem was solved, the next challenge was how to perform gene silencing. Since the addition of the DNA methyltransferase domain DNMT3A by CRISPRoff is cytotoxic, here the researchers came up with another strategy – instead of transporting DNMT3A from the outside, they directly recruit the endogenous DNMT3A of the cells themselves, thus avoiding cytotoxicity while further reducing the load.
DNMT3A is inherently inactive, and the research team then proposed an ingenious strategy to activate DNMT3A at the target site. They combine the companion molecules of DNMT3A and attach them to zinc finger proteins, which reach the target PrP gene, so that endogenous DNMT3A is activated when it encounters the target gene, acting as a silencing gene.
▲CHARM recruits endogenous DNMT3A and activates DNMT3A (Image source: Reference [1])
Finally, this study also solves the off-target problem that previously existed with CRISPRoff. In their design, one of the editors targets the promoter in the AAV vector, so that the novel editor can self-silence the expression of all editors after completing the task of gene silencing, thus avoiding the adverse effects that can result from long-term expression of editing tools.
Long-term inhibition of lethal proteins
The above is a beautiful answer sheet submitted by Vallabh et al. called CHARM (full name is Coupled Histone tail for Autoinhibition Release of Methyltransferase). In mouse experiments, CHARM was able to knock down more than 80% of PrP expression in the whole brain, far exceeding the amplitude of PrP inhibition needed to improve symptoms. It is important to know that in previous studies by Vallabh et al., less than 25% of PrP inhibition has been able to take effect. In addition, the CHARM editor achieved self-silencing after silencing the target gene and did not result in cytotoxicity as well as other adverse effects.
▲Long-term inhibition of PrP was achieved by transient expression of CHARM (Image source: Reference [1])
Commenting on this potentially revolutionary tool, a contemporaneous opinion article commented that the development of CHARM introduces an effective and safe editing technique that can be delivered via AAV to hard-to-target organs such as the brain, thereby achieving gene silencing. While the persistence of gene silencing through methylation remains to be confirmed, and gene silencing may only be useful for some diseases, epigenetic editors such as CHARM could eventually have a dramatic impact on human health similar to tools such as base editing, lead editing, and others.
According to the research team, they are currently adapting the CHARM and modularizing the tool to further improve its effectiveness, safety, and ability to be mass-produced.
From basic research in the laboratory to the final treatment, there is always a long and winding road. For Vallabh, the "hanging sword" has not yet been taken down, and there is no reason to stop in this race against time to save himself and more patients. As Vallabh previously wrote on the Broad Institute website: We will continue to move forward, constantly updating our understanding of "moving forward."
Resources:
[1] Edwin N. Neumann et al., Brain-wide silencing of prion protein by AAV-mediated delivery of an engineered compact epigenetic editor. Science (2024). DOI: 10.1126/science.ado7082
[2] A CHARMed collaboration created a potent therapy candidate for fatal prion diseases. Retrieved June 28, 2024 from https://www.eurekalert.org/news-releases/1048956
[3] Vallabh, S. M. et al. Prion protein quantification in human cerebrospinal fluid as a tool for prion disease drug development. PNAS 201901947 (2019)
[4] Minikel, E. V. et al. Prion protein lowering is a disease-modifying therapy across prion disease stages, strains and endpoints. Nucleic Acids Res. (2020)
[5] James K. Nuñez et al., Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell (2021). DOI: 10.1016/j.cell.2021.03.025