DNA Editing Technique Corrects Defects in RNA-Associated Diseases – R & D Magazine

An updated version of the CRISPR-Cas9 gene editing technique has set its sights on RNA-associated diseases.

Researchers from the University of California San Diego School of Medicine have developed a new technique known as RNA-targeting Cas9 (RCas9) to correct molecular mistakes that can lead to microsatellite repeat expansion diseases including myotonic dystrophy types 1 and 2the most common form of hereditary ALS and Huntingtons disease.

This is exciting because we're not only targeting the root cause of diseases for which there are no current therapies to delay progression but we've re-engineered the CRISPR-Cas9 system in a way that's feasible to deliver it to specific tissues via a viral vector, senior author Gene Yeo, Ph.D., professor of cellular and molecular medicine at UC San Diego School of Medicine, said in a statement.

Microsatellite repeat expansion diseases are causes by errant repeats in RNA sequences that are toxic to the cell because they prevent production of crucial proteins.

Repetitive RNAs will accumulate in the nucleus or cytoplasm of cells to form dense knots called foci.

During the study, the researchers used the new technique to eliminate problem-causing RNAs associated with microsatellite repeat expansion diseases in patient-derived cells and cellular models of the diseases.

Under normal circumstances researchers will design a guide RNA to match the sequence of a specific target gene. The RNA will direct the Cas9 enzyme to the desired spot in the genome to cut the DNA.

The cell repairs the DNA break imprecisely, inactivating the gene. The researchers also can replace the section adjacent to the cut with a corrected version of the gene. RCas9 will work similarly, but the guide RNA directs Cas9 to an RNA molecule instead of DNA.

In the laboratory, the researchers tested the new technique on microsatellite repeat expansion disease RNAs and found that 95 percent or more of the RNA foci linked to myotonic dystrophy type 1 and type 2 one type of ALS and Huntingtons disease were eliminated.

RCas9 also reversed 93 percent of MBNL1a protein that normally binds RNA but is sequestered away from hundreds of its natural RNA targets by the RNA foci in myotonic dystrophy type 1in patient muscles cells and the cells ultimately resembled healthy control cells.

However, challenges must be overcome before RCas9 could be used on patients, as efficient delivery of RCas9 to patient cells is not yet perfected. Non-infectious adeno-associated viruses are commonly used in gene therapy but are too small to hold Cas9 to target DNA.

The researchers developed a smaller version of Cas9 by deleting regions of the protein that were necessary for DNA cleavage but dispensable for binding RNA.

The main thing we don't know yet is whether or not the viral vectors that deliver RCas9 to cells would elicit an immune response, Yeo said. Before this could be tested in humans, we would need to test it in animal models, determine potential toxicities and evaluate long-term exposure.

The study was published in Cell.

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DNA Editing Technique Corrects Defects in RNA-Associated Diseases - R & D Magazine