SPLICER: A Breakthrough in Alzheimer's Gene Therapy

A new gene editing technique – SPLICER that allows cellular machinery to skip sections of genes involved for diseases has been used to minimize the creation of amyloid-beta plaque precursors in a mouse model of Alzheimer’s disease, according to researchers at the University of Illinois Urbana-Champaign.

The application in living mice demonstrates the technique, called SPLICER,’s improved efficiency over the current standard in gene editing technology, as well as its potential for use in other disorders, the researchers said. 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 useful for health disorders caused by mutations that result in misfolded or toxic proteins, such as Duchenne’s muscular dystrophy or Huntington disease.

“DNA contains the instructions to build everything that is responsible for how cells function. So it’s like a book of recipes that contains very detailed instructions for cooking,” Perez-Pinera said.

But there are large regions of DNA that don’t code for anything. It’s like, you start the recipe for a turkey dinner, and then you hit a note that says, ‘continued on page 10.’ After page 10, it’s ‘continued on page 25.’ The pages between are gibberish.”

Pablo Perez-Pinera, Professor, Bioengineering, University of Illinois at Urbana-Champaign

“But say on one of the recipe pages -; in genetics, an exon -; there is a typo that makes the turkey inedible, or even poisonous. If we cannot correct the typo directly, we could amend the note before it to send you to the next page, skipping over the page with the error, so that at the end you could make an edible turkey. Though you might lose out on the gravy that was on the skipped page, you’d still have dinner. In the same way, if we can skip the piece of the gene with the toxic mutation, the resulting protein could still have enough function to perform its critical roles.”

SPLICER extends the popular CRISPR-Cas9 gene editing tool with significant improvements. CRISPR-Cas9 systems require a certain DNA sequence to latch onto, which limits the genes that can be altered. SPLICER employs newer Cas9 enzymes that do not require that sequence, opening the door to additional targets such as the Alzheimer’s-related gene that the Illinois researchers focused on.

“Another problem we address in our work is precision in what gets skipped,” said graduate student Angelo Miskalis, a co-first author of the paper. “With current exon-skipping techniques, sometimes not all of the exon gets skipped, so there’s still part of the sequence we don’t want expressed. In the cookbook analogy, it’s like trying to skip a page, but the new page starts in the middle of a sentence, and now the recipe doesn’t make sense. We wanted to prevent that.”

There are two important sequence sections surrounding an exon that inform the cellular machinery which parts of a gene to use for protein synthesis: one at the start and one at the end. Most exon-skipping technologies only target one sequence, while SPLICER changes both the beginning and end sequences. As a result, Miskalis claims that the targeted exons are skipped more efficiently.

The Illinois researchers chose to target an Alzheimer’s gene for the first demonstration of SPLICER’s therapeutic powers because, while the target gene has been extensively investigated, efficient exon skipping in living animals has remained elusive. The researchers focused on a specific exon coding for an amino acid sequence inside a protein that is cleaved to generate amyloid-beta, which accumulates to form plaques on neurons in the brain as diasease progresses.

SPLICER significantly reduced amyloid-beta production in cultured neurons. When the researchers examined the DNA and RNA production of mouse brains, they discovered that the targeted exon was reduced by 25% in the SPLICER-treated mice, with no evidence of off-target effects.

“When we originally tried to target this exon with older techniques, it didn’t work,” said graduate student Shraddha Shirguppe, also a co-first author of the study. “Combining the newer base editors with dual splice editing skipped the exon at a much better rate than we were previously able to with any of the available methods. We were able to show that not only could it skip the whole exon better, it reduced the protein that produces the plaque in these cells.”

“Exon skipping only works if the resulting protein is still functional, so it can’t treat every disease with a genetic basis. That’s the overall limitation of the approach,” Perez-Pinera said. “But for diseases like Alzheimer’s, Parkinson’s, Huntington’s or Duchenne’s muscular dystrophy, this approach holds a lot of potential. The immediate next step is to look at the safety of removing the targeted exons in these diseases, and make sure we aren’t creating a new protein that is toxic or missing a key function. We would also need to do longer term animal studies and see if the disease progresses over time.”

Source: University of Illinois at Urbana-Champaign