Researchers at the University of California at San Diego have used RNA-targeted CRISPR to reverse symptoms in an animal model of myotonic dystrophy type 1 (DM1). They reported their findings in the Sept. 14, 2020, issue of Nature Biomedical Engineering.

Microsatellite repeat expansions, or repeat sequences of a few base pairs length, can be toxic in multiple different ways. In some cases, the DNA repeats code for amino acids, leading to proteins with extra amino acids that cause them to misfold and aggregate. There is a whole family of polyglutamine (polyQ) diseases, most famously Huntington’s disease, that are caused by a repeat expansion that codes for glutamine.

In DM1, the toxicity mechanisms are different.

The repeat expansion is in the noncoding part of the RNA for myotonic dystrophy protein kinase (DMPK), and rather than producing a dysfunctional protein, the RNA repeat acts as a “sponge” of sorts, Gene Yeo explained, trapping RNA-binding proteins. That in turns leads to “global RNA splicing dysfunction in the cells,” he told BioWorld.

Destroying the RNA with repeat expansions restores about 85% to 90% of normal splicing. And because only one copy of the gene has a long repeat expansion, DMPK that can carry out its normal enzymatic function is still being produced off the other copy.

Yeo is a professor of cellular and molecular medicine at UC San Diego School of Medicine, and the senior author of the paper reporting the results.

His lab is focused on adapting the gene editing technology CRISPR, which normally targets DNA, to RNA instead.

Using RNA-targeted CRISPR to go after the expansions has advantages over other RNA-targeting approaches, he said.

To be effective, short interfering RNA (siRNA) needs to activate the protein argonaute, which is found in the cytoplasm. But many RNAs with repeat expansion remain trapped in the nucleus.

Antisense oligonucleotides (ASOs) have empirically been tough to deliver into muscle cells, and do not appear to bind the repeats very strongly.

Yeo and his team had previously established proof of concept of the RNA-targeting CRISPR in cell lines, “so we were quite comfortable with the mechanistic part,” he said. “But we were worried about immunogenic responses” to either the Cas protein or the viral vector.

However, when they tested their approach in newborn mice, the animals “built tolerance very quickly to the foreign protein,” showing that the approach could be feasible to treat babies with early-onset cases.

But adult mice, too, tolerated the treatment well if the team co-treated them with two weeks of initial immunosuppression.

The researchers did see some activated T cells, but after two weeks of immune suppression similar to that used for organ transplants, “mice did not demonstrate overt immunity that caused muscle damage,” Yeo said.

He acknowledged that even after the fact, “I don’t have the full explanation” for why there was only a minimal immune response in the adult animals, but noted that he and his colleagues used doses of virus that were several orders of magnitude lower than those that have been used in other studies.

In the work now published in Nature Biomedical Engineering, treating animals with a single injection of the RNA-targeted CRISPR system resulted in “a restorative effect on molecular, cellular and behavioral aspects of the disease,” he said.

Yeo is co-founder and scientific advisory board chair at Locanabio Inc., which focuses on developing RNA-targeting gene therapy using CRISPR targeting as well as other proteins. The company was founded in 2016 and had a series A financing in 2019. Locanabio is planning to move the approach into large animal models.

On the scientific side, Yeo’s team is interested in looking at another aspect of DM1 that occurs in the congenital, more severe form of the disease, where the RNA repeats can number in the thousands.

The symptoms of congenital DM1 include “a very dramatic CNS problem” in affected infants, Yeo said, which is puzzling because the proteins that seem to be most affected in DM1 are not expressed in the brain. “So we are very interested in that biologically.”

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