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 report. the University of Illinois at Urbana-Champaign.
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 Pérez-Piñera, 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 muscular dystrophy or Huntington’s disease.
“DNA contains the instructions to build everything that is responsible for the functioning of cells. So it is like a recipe book that contains very detailed instructions for cooking,” Pérez-Piñera 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 ‘continue on page 10.’ After page 10, “continue on page 25 “. The middle pages are gibberish.
“But let’s say that on one of the recipe pages (in genetics, an exon) there is a typo that makes the turkey inedible or even poisonous. If we can’t correct the typo directly, we could modify the above note to send you to the next page, skipping the page with the error, so that in the end you can make an edible turkey. Although you might lose the sauce that was on the skipped page, you would still have dinner in the same way, if we “Can skip the part of the. gene with toxic mutation, the resulting protein could still have enough function to perform its critical functions.”
SPLICER is based on the popular CRISPR-Cas9 gene editing platform, with key changes. CRISPR-Cas9 systems require a specific DNA sequence to attach, limiting which genes can be edited. SPLICER uses newer Cas9 enzymes that don’t need that sequence, opening the door to new targets like the Alzheimer’s-related gene the Illinois group focused on.
“Another issue we address in our work is precision in what is omitted,” said graduate student Angelo Miskalis, co-author of the paper. “With current exon skipping techniques, sometimes the entire exon is not skipped, so there is still part of the sequence that we don’t want to express. 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.
There are two key sequence areas surrounding an exon that tell the cellular machinery which parts of a gene to use to make proteins: one at the beginning and one at the end. While most exon skipping tools target only one sequence, SPLICER edits both the start and end sequences. As a result, targeted exons are skipped more efficiently, Miskalis said.
The Illinois group chose to target an Alzheimer’s gene for the first demonstration of SPLICER’s therapeutic capabilities because, while the target gene has been well studied, efficient exon skipping remains elusive in living organisms. The researchers targeted a specific exon that codes for an amino acid sequence within a protein that is cleaved to form beta-amyloid, which builds up to form plaques in brain neurons as the disease progresses.
In cultured neurons, SPLICER effectively reduced beta-amyloid formation. By analyzing DNA and RNA production from mouse brains, the researchers found that the targeted exon was decreased by 25% in mice treated with SPLICER, 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-author of the study. “Combining the newer base editors with dual splicing editing skipped the exon at a much better rate than we could previously achieve with any of the available methods. We were able to show that not only could it skip the entire exon better, but “It also reduced the protein that plaque produces in these cells.”
“Exon skipping only works if the resulting protein is still functional, so it cannot treat all diseases with a genetic basis. That is the general limitation of the approach,” Pérez-Piñera said. “But for diseases like Alzheimer’s, Parkinson’s, Huntington’s muscular dystrophy or Duchenne muscular dystrophy, this approach has great potential. The next immediate step is to analyze the safety of deleting specific exons in these diseases and make sure that We are not creating “a new protein that is toxic or lacks a key function. “We would also need to do longer-term animal studies and see if the disease progresses over time.”
At Illinois, Pérez-Piñera is also affiliated with the department of Molecular and Integrative Physiology, the Carle Illinois College of Medicine, the Illinois Cancer Center, and the Carl R. Woese Institute for Genomic Biology. U. of I. Bioengineering professors Sergei Maslov and Thomas Gaj were co-authors 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.
This work was supported by National Institutes of Health grants 1U01NS122102, 1R01NS123556, 1R01GM141296, 1R01GM127497, T32EB019944, and 1R01GM131272.