Revolutionary Gene Editing Technique Slashes Alzheimer’s Plaque Precursor in Mice

This application in living mice highlights the enhanced effectiveness of the tool, named SPLICER, compared to existing gene editing technologies, as well as its potential for use in other medical conditions, said the researchers. The study, led by Pablo Perez-Pinera, a bioengineering professor at the University of Illinois, was published in the journal Nature Communications.

SPLICER employs a gene editing technique known as exon skipping, which is particularly relevant for health issues stemming from mutations that create misfolded or harmful proteins, including Duchenne’s muscular dystrophy and Huntington’s disease.

“DNA holds the blueprints for constructing everything that governs cellular function. It’s analogous to a recipe book filled with elaborate instructions for cooking,” Perez-Pinera explained. “Yet, significant portions of DNA do not code for anything meaningful. It’s like starting a turkey dinner recipe only to hit a note saying, ‘continued on page 10.’ After that, it goes on to ‘continued on page 25.’ The in-between is nonsensical.

“Imagine there’s a typo on one of the essential recipe pages — in genetics, an exon — that renders the turkey inedible or even harmful. If we can’t fix that typo directly, we can modify the prior instruction to jump ahead, bypassing the erroneous section, ultimately allowing for an edible turkey at the end. While you might lose out on some gravy from the skipped portion, you’d still have a meal. Similarly, if we can exclude the part of the gene with the harmful mutation, the resulting protein may still retain enough functionality to fulfill its vital tasks.”

Building on the widely-used CRISPR-Cas9 gene editing system, SPLICER incorporates significant improvements. Traditional CRISPR-Cas9 methods require a specific DNA sequence to bind, which restricts the range of editable genes. In contrast, SPLICER utilizes advanced Cas9 enzymes that do not require such a sequence, thereby expanding the possibilities for targeting, including the Alzheimer’s-related gene the Illinois researchers investigated.

“Another challenge we tackle in our work is ensuring precision in what is skipped,” remarked Angelo Miskalis, a graduate student and co-first author of the paper. “Existing exon-skipping methods often struggle with skipping the entire exon, leaving unwanted parts of the sequence expressed. In our cookbook analogy, it’s like trying to circumvent a page only to find that the new page starts midway through a sentence, making the recipe nonsensical. We aimed to avoid that issue.”

There are two crucial sequence regions flanking an exon that guide the cellular machinery on which gene parts to utilize for protein synthesis: one at the start and the other at the end. Most exon-skipping techniques only target one of these sequences, whereas SPLICER modifies both the beginning and ending sequences. This improvement leads to more efficient skipping of the intended exons, as Miskalis indicated.

The Illinois team chose to focus on an Alzheimer’s gene for the inaugural demonstration of SPLICER’s therapeutic potential because, although this gene has been extensively studied, effective exon skipping in living organisms has been challenging to achieve. They targeted a specific exon that encodes an amino acid sequence within a protein that gets cleaved, resulting in amyloid-beta, which builds up to create plaques on neurons as the disease progresses.

In cultured neurons, SPLICER significantly curtailed amyloid-beta formation. Upon analyzing the DNA and RNA outputs from mouse brains, the researchers discovered that the targeted exon was decreased by 25% in the mice treated with SPLICER, with no indications of off-target effects.

“When we initially attempted to target this exon with older methods, it was unsuccessful,” stated Shraddha Shirguppe, a graduate student and co-first author of the study. “Integrating the newer base editors with dual splice editing enabled us to skip the exon more effectively than with any previously available techniques. We demonstrated not only that we could skip the complete exon more efficiently, but also that it diminished the protein responsible for plaque formation in these cells.”

“Exon skipping is only effective if the resulting protein continues to function, limiting its application for all genetically based diseases. This is the fundamental constraint of the method,” Perez-Pinera noted. “However, for conditions like Alzheimer’s, Parkinson’s, Huntington’s, or Duchenne’s muscular dystrophy, this technique offers significant promise. The next immediate step is to assess the safety of removing the targeted exons in these conditions to ensure that we are not creating a new protein that is either toxic or lacks a key function. Additionally, long-term animal studies are necessary to determine if the disease progresses over time.”

At the University of Illinois, Perez-Pinera is also associated with the Department of Molecular and Integrative Physiology, the Carle Illinois College of Medicine, the Cancer Center at Illinois, and the Carl R. Woese Institute for Genomic Biology. Coauthors on the paper include U. of I. Bioengineering professors Sergei Maslov and Thomas Gaj. This research was supported by various organizations, including the National Institutes of Health, the Muscular Dystrophy Association, the American Heart Association, the Parkinson’s Disease Foundation, and the Simons Foundation.

The funding for this work was provided through National Institutes of Health grants 1U01NS122102, 1R01NS123556, 1R01GM141296, 1R01GM127497, T32EB019944, and 1R01GM131272.