RNA-targeting CRISPR System Shows Promise in Preclinical Models

Marisa Wexler, MS avatar

by Marisa Wexler, MS |

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A CRISPR-based gene editing system could be used to reduce the activity of genes associated with amyotrophic lateral sclerosis (ALS) and Huntington’s disease, a new study shows.

The study, “Targeted gene silencing in the nervous system with CRISPR-Cas13,” was published in Science Advances.

CRISPR is a strategy that bacteria evolved to fight off viruses and, in recent years, the system has gained widespread research interest as a tool for gene editing. Very simply, CRISPR involves a small piece of RNA called a “guide RNA,” with a specific sequence that lets it target a particular gene. The guide RNA signals a protein called a Cas enzyme to destroy or alter the targeted gene.

Much CRISPR research has specifically used the Cas9 enzyme, which targets DNA. However, this poses some safety risks — an accidental change in a cell’s DNA can fundamentally alter cellular activity and may contribute to developing cancer.

When a gene is “read,” cells make a temporary molecule called messenger RNA (mRNA) to carry the genetic code from the DNA to the cell’s protein-making machines, called ribosomes. Theoretically, reducing mRNA levels of a disease-driving gene could be a viable therapeutic strategy in diseases with known genetic drivers. For instance, mutations in a gene called SOD1 contribute to many ALS cases.

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“Targeting RNA rather than DNA has some unique advantages, including the fact that, in theory, its effects within a cell can be reversed since RNAs are transient molecules,” Colin Lim, a graduate student at the University of Illinois Urbana-Champaign and co-author of the study, said in a news story.

In the study, Lim and other scientists at the University of Illinois designed a system to target SOD1 with a CRISPR-based system that uses a Cas enzyme called Cas13 (specifically a version called RfxCas13d), which targets RNA instead of DNA.

“Because Cas13 enzymes just target RNA, they also carry minimal risk for introducing any permanent off-target mutations to DNA,” Lim said.

The team designed a system that uses an adeno-associated virus (AAV) to deliver the Cas13 enzyme alongside a guide RNA targeting SOD1 mRNA to astrocytes, a particular type of nervous system cell known to be involved in SOD1-associated ALS. AAV is a virus that doesn’t cause disease in people, and often is used in gene therapy studies because it is easy to manipulate in a laboratory.

After refining the process in cellular models, the researchers tested their system in a mouse model of SOD1-associated ALS and showed that treating mice with the CRISPR-Cas13 system didn’t alter disease onset, which was expected, since onset is only delayed when the SOD1 protein produced by the defective gene is lowered in motor neurons.

However, treated mice lived significantly longer and had a slower disease course than control mice, as evidenced by a 33% increase in the time between disease onset and end-stage disease. Treated mice also performed better on motor function tests during the late stage of disease, and muscle atrophy was about three times slower in treated mice over the course of the disease.

Analyses of the mice’s tissue confirmed that the treatment significantly reduced SOD1 activity, without triggering inflammatory responses against the CRISPR-Cas13 components.

“Our results demonstrate that RfxCas13d can be programmed to target SOD1 and that its AAV-mediated delivery to the spinal cord can impart a therapeutic benefit to a mouse model of SOD1-linked ALS,” the researchers concluded.

In other experiments, the researchers showed that the same system could be applied to target the HTT gene mutated in Huntington’s disease. They also showed that the Cas13 protein was not targeting unintended genes.

“In our experiments, Cas13 was generally as specific as the more established gene-silencing modalities that we tested alongside of it, but determining exactly how specific — and thus how safe — Cas13 is in human cells remains a critical question for the field,” Thomas Gaj, PhD, a professor at Illinois and co-author of the study, said.

“We are excited about exploring the potential of Cas13 and other RNA-targeting CRISPR enzymes. However, the technology is still in its infancy,” Gaj added. “Many important questions about its specificity and its ability to cleave non-target RNAs need to be answered, all of which will help guide its refinement and its future use.”