(maybe something for prion diseases...)
(...)
“CHARMs are an elegant solution to the problem of silencing disease genes, and they have the potential to have an important position in the future of genetic medicines.”
To treat a genetic disease, target the gene
Prion disease, which leads to swift neurodegeneration and death, is caused by the presence of misshapen versions of the prion protein. These cause a cascade effect in the brain: the faulty prion proteins deform other proteins, and together these proteins not only stop functioning properly but also form toxic aggregates that kill neurons. The most famous type of prion disease, known colloquially as mad cow disease, is infectious, but other forms of prion disease can occur spontaneously or be caused by faulty prion protein genes.
Most conventional drugs work by targeting a protein. CHARMs, however, work further upstream, turning off the gene that codes for the faulty protein so that the protein never gets made in the first place. CHARMs do this by epigenetic editing, in which a chemical tag gets added to DNA in order to turn off or silence a target gene. Unlike gene editing, epigenetic editing does not modify the underlying DNA — the gene itself remains intact. However, like gene editing, epigenetic editing is stable, meaning that a gene switched off by CHARM should remain off. This would mean patients would only have to take CHARM once, as opposed to protein-targeting medications that must be taken regularly as the cells’ protein levels replenish.
Research in animals suggests that the prion protein isn’t necessary in a healthy adult, and that in cases of disease, removing the protein improves or even eliminates disease symptoms. In a person who hasn’t yet developed symptoms, removing the protein should prevent disease altogether. In other words, epigenetic editing could be an effective approach for treating genetic diseases such as inherited prion diseases. The challenge is creating a new type of therapy.
Fortunately, the team had a good template for CHARM: a research tool called CRISPRoff that Weissman’s group previously developed for silencing genes. CRISPRoff uses building blocks from CRISPR gene editing technology, including the guide protein Cas9 that directs the tool to the target gene. CRISPRoff silences the targeted gene by adding methyl groups, chemical tags that prevent the gene from being transcribed, or read into RNA, and so from being expressed as protein. When the researchers tested CRISPRoff’s ability to silence the prion protein gene, they found that it was effective and stable.
Several of its properties, though, prevented CRISPRoff from being a good candidate for a therapy. The researchers’ goal was to create a tool based on CRISPRoff that was just as potent but also safe for use in humans, small enough to deliver to the brain, and designed to minimize the risk of silencing the wrong genes or causing side effects.
From research tool to drug candidate
Led by Neumann and Bertozzi, the researchers began engineering and applying their new epigenome editor. The first problem that they had to tackle was size, because the editor needs to be small enough to be packaged and delivered to specific cells in the body. Delivering genes into the human brain is challenging; many clinical trials have used adeno-associated viruses (AAVs) as gene-delivery vehicles, but these are small and can only contain a small amount of genetic code. CRISPRoff is way too big; the code for Cas9 alone takes up most of the available space.
The Weissman lab researchers decided to replace Cas9 with a much smaller zinc finger protein (ZFP). Like Cas9, ZFPs can serve as guide proteins to direct the tool to a target site in DNA. ZFPs are also common in human cells, meaning they are less likely to trigger an immune response against themselves than the bacterial Cas9.
Next, the researchers had to design the part of the tool that would silence the prion protein gene. At first, they used part of a methyltransferase, a molecule that adds methyl groups to DNA, called DNMT3A. However, in the particular configuration needed for the tool, the molecule was toxic to the cell. The researchers focused on a different solution: Instead of delivering outside DNMT3A as part of the therapy, the tool is able to recruit the cell’s own DNMT3A to the prion protein gene. This freed up precious space inside of the AAV vector and prevented toxicity.
The researchers also needed to activate DNMT3A. In the cell, DNMT3A is usually inactive until it interacts with certain partner molecules. This default inactivity prevents accidental methylation of genes that need to remain turned on. Neumann came up with an ingenious way around this by combining sections of DNMT3A’s partner molecules and connecting these to ZFPs that bring them to the prion protein gene. When the cell’s DNMT3A comes across this combination of parts, it activates, silencing the gene.
“From the perspectives of both toxicity and size, it made sense to recruit the machinery that the cell already has; it was a much simpler, more elegant solution,” Neumann says. “Cells are already using methyltransferases all of the time, and we’re essentially just tricking them into turning off a gene that they would normally leave turned on.”
Testing in mice showed that ZFP-guided CHARMs could eliminate more than 80 percent of the prion protein in the brain, while previous research has shown that as little as 21 percent elimination can improve symptoms.
Once the researchers knew that they had a potent gene silencer, they turned to the problem of off-target effects. The genetic code for a CHARM that gets delivered to a cell will keep producing copies of the CHARM indefinitely. However, after the prion protein gene is switched off, there is no benefit to this, only more time for side effects to develop, so they tweaked the tool so that after it turns off the prion protein gene, it then turns itself off.
Meanwhile, a complementary project from Broad Institute scientist and collaborator Benjamin Deverman’s lab, focused on brain-wide gene delivery and published in Science on May 17, has brought the CHARM technology one step closer to being ready for clinical trials. Although naturally occurring types of AAV have been used for gene therapy in humans before, they do not enter the adult brain efficiently, making it impossible to treat a whole-brain disease like prion disease. Tackling the delivery problem, Deverman’s group has designed an AAV vector that can get into the brain more efficiently by leveraging a pathway that naturally shuttles iron into the brain. Engineered vectors like this one make a therapy like CHARM one step closer to reality.
(more)
https://news.mit.edu/2024/charmed-collaboration-creates-therapy-candidate-fatal-prion-diseases-0627....
Brainwide silencing of prion protein by AAV-mediated delivery of an engineered compact epigenetic editor
https://www.science.org/doi/10.1126/science.ado7082Prion diseases are devastating neurodegenerative disorders that are invariably fatal, but removal of the prion protein from neurons can protect against disease progression. Neumann et al. developed a compact epigenetic silencer called CHARM that could efficiently shut off the prion gene throughout the mouse brain when delivered systemically by a viral vector without changing the underlying DNA sequence (see the Perspective by Whittaker and Musunuru). The epigenetic editor can also be programmed to turn itself off after silencing its target, thus limiting potential adverse effects from long-term expression. CHARM represents a therapeutic modality that could be applied to a range of other diseases caused by the toxic buildup of unwanted proteins. —Di Jiang
Structured Abstract
INTRODUCTION
Prion diseases are fatal neurodegenerative disorders caused by misfolding of the prion protein in the brain. Cases can manifest spontaneously, be inherited genetically, or be acquired through transmission (e.g., mad cow disease). Although there are currently no effective treatments, reducing prion protein levels in the brain has been shown to halt disease progression in animal models with minimal adverse effects. In addition, prion protein is nonessential in mammals, indicating that lowering its expression in the brain is a viable therapeutic strategy.
RATIONALE
Genetic medicines hold great promise but are often difficult to translate to the clinic. Current CRISPR-based DNA-editing technologies are complex large molecules that are challenging to deliver and have been associated with unintended editing outcomes. We therefore favored an epigenetic editing approach to permanently turn off prion protein expression in the brain without altering the underlying DNA sequence or leading to continued expression of an altered mRNA and protein. This strategy uses DNA methylation to achieve long-term transcriptional silencing. However, current epigenetic editors are cytotoxic in some circumstances and are too large to fit in an adeno-associated virus (AAV) vector, the preferred delivery vehicle to the central nervous system.
RESULTS
To address these challenges, we engineered a compact, enzyme-free epigenetic editor termed CHARM (Coupled Histone tail for Autoinhibition Release of Methyltransferase). Through a direct fusion with the histone H3 tail and a noncatalytic Dnmt3l domain, CHARM is able to recruit and activate DNA methyltransferases endogenously expressed in the cell to methylate the target gene. CHARM can act independently of KRAB transcriptional repression domains and is compatible with multiple DNA-binding modalities, including CRISPR-Cas, transcription activator–like effectors, and zinc finger proteins. The small size of zinc finger proteins enables up to three DNA targeting elements to be accommodated in a single AAV with additional room for regulatory elements to confer cell-type specificity. When coupled to a prion protein–targeting zinc finger domain and delivered to the mouse brain through AAV, CHARM methylates the prion gene promoter and achieves up to 80% brainwide reduction in neuronal prion protein, far exceeding the minimal reduction required for therapeutic benefit. Furthermore, we developed self-silencing CHARMs that autonomously deactivate themselves after silencing their target. This approach temporally limits CHARM expression to circumvent potential antigenicity and off-target activity resulting from chronic expression in nondividing neurons.
CONCLUSION
This study represents the first demonstration of AAV-mediated delivery of an epigenetic editor that can programmably methylate DNA in the brain for durable, potent silencing of a target gene. CHARM avoids overexpression of potentially cytotoxic catalytic domains by harnessing the endogenous DNA methylation machinery. Its compact size enables modular self-silencing strategies, facilitates multiplexed targeting, and enhances compatibility with other delivery modalities, such as lipid nanoparticles. This work could enable an effective treatment for patients with prion disease as well as other neurodegenerative diseases involving the accumulation of toxic protein aggregates. More generally, CHARM represents the next generation of safe and easily deliverable epigenetic editors for therapeutic intervention and biological discovery.
(more)
