SPLICER shows promise in Alzheimer's and other diseases

A new gene editing tool that helps cellular machinery skip parts of genes responsible for diseases has been applied to reduce the formation of amyloid-beta plaque precursors in a mouse model of Alzheimer's disease, researchers at the University of Illinois Urbana-Champaign report. 

The application in live mice shows the improved efficiency of the tool, called SPLICER, over the current standard in gene editing technology, as well as the potential for application in other diseases, the researchers said. Led by Pablo Perez-Pinera, a professor of bioengineering at the U. of I., the researchers published their findings in the journal Nature Communications. 

SPLICER uses a gene editing approach called exon skipping, which is of particular interest for health conditions caused by mutations that produce misfolded or toxic proteins, such as Duchenne's muscular dystrophy or Huntington's 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 builds upon the popular CRISPR-Cas9 gene editing platform -; with key changes. CRISPR-Cas9 systems require a specific DNA sequence to latch on, limiting which genes could be edited. SPLICER uses newer Cas9 enzymes that do not need that sequence, opening up the door to new targets like the Alzheimer's-related gene that the Illinois group 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 key sequence areas surrounding an exon that tell the cellular machinery which parts of a gene to use for making proteins: one at the beginning and one at the end. While most exon-skipping tools target only one sequence, SPLICER edits both the starting and ending sequences. As a result, the targeted exons are skipped over more efficiently, Miskalis said. 

The Illinois group chose to target an Alzheimer's gene for the first demonstration of SPLICER's therapeutic abilities because while the target gene has been well-studied, efficient exon skipping has remained elusive in living organisms. The researchers targeted a specific exon coding for an amino acid sequence within a protein that gets cleaved to form amyloid-beta, which accumulates to form plaques on neurons in the brain as the disease progresses. 

In cultured neurons, SPLICER reduced the formation of amyloid-beta efficiently. When analyzing the DNA and RNA output of mouse brains, the researchers found that the targeted exon was decreased 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."

At Illinois, Perez-Pinera also is affiliated 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. U. of I. Bioengineering professors Sergei Maslov and Thomas Gaj were coauthors of the paper. The National Institutes of Health, the Muscular Dystrophy Association, the American Heart Association, the Parkinson's Disease Foundation and the Simons Foundation supported this work.