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UT Southwestern Team Publishes Details on Duplex RNA Inhibition of Huntington's Disease Gene

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Researchers from the University of Texas Southwestern Medical Center this month published new data describing the mechanism by which mismatch-containing double-stranded RNAs preferentially inhibit the mutant gene that causes Huntington's disease over the wild-type gene.

While much work remains before such molecules can be used to treat Huntington's disease, the findings support their continued development as a more selective alternative to single-stranded antisense approaches being advanced by others, according to UT Southwestern's David Corey, the study's senior author.

Huntington's disease is a neurodegenerative condition caused by expanded CAG trinucleotide repeats within the huntingtin gene, which results in the production of a toxic mutant protein. Given its genetic nature, the disease has been a popular target for those developing RNA-based gene-silencing drugs, including Alnylam Pharmaceuticals, although that company dropped out of a Huntington's disease collaboration earlier this year to focus on other pipeline programs (GSN 5/10/2012), and Isis Pharmaceuticals.

Also interested in applying RNAi to the disease is Corey, who in 2009 reported in Nature Biotechnology on an allele-specific strategy to silence mutant huntingtin.

Noting that the majority of RNAi and antisense agents targeting huntingtin inhibit both the mutant and wild-type versions of the protein, which raises concerns of potentially significant side effects, Corey and his colleagues aimed to target only the mutant form using single-stranded antisense oligos.

“Triplet repeat sequences within RNA can form hairpin structures,” they wrote. Because the expanded repeats of mutant huntingtin “create additional target sequence and more potential binding sites,” they designed their antisense molecules against CAG repeats.

They found that, in vitro, the antisense agents “could generate from four- to better than eight-fold selectivity for the mutant versus the wild-type,” Corey told Gene Silencing News this week. “We also reported that double-stranded RNAs were not selective at all.”

The next year, Corey's lab published a paper showing that duplex RNAs, when switched away from a traditional RNAi mechanism toward an miRNA-like one through the introduction of one or more mismatched bases, could potently and selectively inhibit mutant huntingtin in patient-derived cells.

“We reasoned that the lack of selectivity [of traditional dsRNAs] might have been because the fully complementary ones were just too powerful and couldn't discriminate the subtle differences between the mutant and wild-type expanded repeats,” he explained.

Though the mismatch-containing duplex RNAs “worked beautifully,” the paper did not explore the mechanism behind the results, Corey said. The latest paper, which appeared in Nucleic Acids Research. does just that.

In it, Corey's team showed that Argonaute 2-mediated recognition of huntingtin mRNA is “the first step in the mechanism of allele-selective silencing. … Because the expanded repeat offers multiple binding sites, more than one anti-CAG RNA complex can bind to each mRNA repeat simultaneously.

They also demonstrated that GW182/TNRC6, a protein family that interacts with Ago2, is recruited to the expanded CAG repeat and that “that there is very strong evidence for cooperativity in these interactions,” Corey noted, “which supports a model where multiple of these small RNAs are binding to the expanded repeat target.”

“Cooperative binding of multiple mismatched RNAs may be necessary to create a complex that binds strongly enough to disrupt translation of the mutant allele,” the researchers wrote in Nucleic Acids Research.

As such, “chemical modifications should be chosen to retain or enhance the potential for cooperative interactions,” they concluded. “This improved understanding of mechanism should facilitate prioritizing the selection of duplex RNAs and expedite drug design and development.”

Corey noted this week that single-stranded RNA approaches to treat Huntington's disease are more advanced than ones based on duplex RNAs, and that Isis has generated compelling data that indicates a clinical candidate might not be too far off.

Most notably, Isis, in collaboration with Corey and the University of California, San Diego's Don Cleveland, published data in Cell this summer showing that single-stranded RNAi oligos could very potently inhibit mutant huntingtin in patient-derived cells.

In line with Corey's previous findings, the Isis effort determined that “strategic placement of mismatched bases mimics microRNA recognition and optimizes discrimination between mutant and wild-type alleles,” and that the oligos function through the RNAi pathway.

Importantly, intraventricular infusion of the single-stranded siRNA produced selective silencing of the mutant huntingtin allele throughout the brain in a mouse model of the disease, according to the paper. Additionally, there was indication that “even a short period of inhibition would be enough to reverse the course of the disease and halt symptoms,” Corey said.

“The concept is that these diseases are caused by aggregated proteins,” he explained. “If you catch this aggregation early enough … you can reverse the size of the aggregates, maybe get rid of them, and that turns the clock back on the disease.

“That would be very hopeful for patients,” he added. “You'd treat them before they start having severe symptoms, try to get rid of these aggregates, and then the disease doesn't progress.”

Despite the promise of the single-stranded oligo approach, Corey said that further work on duplex RNA methods are warranted, even ones that don't discriminate between mutant and wild-type huntingtin.

“This is a disease with no cures, [and] there are really no other good strategies on the horizon for producing anything that could even approach a cure,” he said. “So there is no reason to put all your eggs in one basket.”

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