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Biological Chemistry

Mismatch Route To Targeted Therapy

RNA with less than perfect complementarity blocks expression of Huntington’s Disease gene sequence

by Stu Borman
November 29, 2010 | A version of this story appeared in Volume 88, Issue 48

Gene Therapy
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Credit: Courtesy of Jiaxin Hu
A fully complementary siRNA complex binds to and induces cleavage of both normal and mutated huntingtin mRNA (left). Mismatched siRNA does not induce cleavage of either type of mRNA and suppresses expression of mutated huntingtin protein selectively—possibly by blocking ribosomal translation.
Credit: Courtesy of Jiaxin Hu
A fully complementary siRNA complex binds to and induces cleavage of both normal and mutated huntingtin mRNA (left). Mismatched siRNA does not induce cleavage of either type of mRNA and suppresses expression of mutated huntingtin protein selectively—possibly by blocking ribosomal translation.

A new therapeutic strategy for treating Huntington’s disease patients that is based on small interfering RNA (siRNA) could have fewer side effects than earlier approaches (Chem. Biol., DOI: 10.1016/j.chembiol.2010.10.013). The technique uses designed synthetic RNAs that behave counterintuitively: They work better when modified to interact less efficiently with their intended targets.

If it can be replicated in vivo, the work could lead to therapies for Huntington’s disease and similar conditions that result from genetic mutations called expansions—elongated repetitive sequences that aren’t found in normal genes.

Huntington’s disease arises from the presence of tens to more than 100 extra copies of a cytosine-adenine-guanine codon in the mutant form of the gene for huntingtin protein. Patients have two copies—or alleles—of the gene, one of which is mutated. The condition causes progressive neurodegeneration that leads to loss of muscle control, mental problems, and, ultimately, death of the patient. No cure exists yet, and effective therapies are urgently needed.

Up to now, efforts to develop therapeutics for the disease have focused on suppressing expression of mutated huntingtin protein in transgenic mice. But agents that reduce levels of the mutated protein also tend to suppress the normal one encoded by the nonmutated allele. This lack of discrimination could lead to adverse side effects in people.

To sidestep this problem, researchers have developed siRNAs that recognize and bind selectively to single-nucleotide polymorphisms (SNPs), genetic signatures that sit outside the huntingtin expansion sequence and serve as markers for the mutant gene. siRNAs inhibit expression of gene transcripts that they complement. Other researchers have suppressed expression of the mutant gene by using siRNAs or antisense oligonucleotides that recognize and bind nonselectively to normal or mutant huntingtin messenger RNA.

It is possible that current generations of allele-selective siRNAs or non-allele-selective siRNAs or antisense oligonucleotides might be adequate as Huntington’s therapies, says chemical biologist David R. Corey of the University of Texas Southwestern Medical Center at Dallas, whose lab developed the new counterintuitive strategy. “I hope they are adequate because they would be the quickest approach to helping patients,” he says. “However, given that these agents will need to be administered into the human brain, possibly chronically, efficient allele-selective strategies are important as second-generation compounds.”

The new strategy, which Corey developed with Jiaxin Hu and Jing Liu, uses siRNAs to achieve selectivity for mutant huntingtin protein by targeting mRNA with the mutated huntingtin expansion sequence. But instead of just designing siRNAs with perfect complementarity to that sequence, Corey and coworkers created siRNAs with intentional sequence mismatches.

Fully complementary siRNA induces cleavage of both normal and mutated huntingtin mRNA. Lack of complementarity in the center of the sequence of siRNA prevents it from inducing cleavage of either type.

The mismatched siRNA has little impact on expression of normal huntingtin protein. But mismatched siRNA does bind to mutated huntingtin mRNA and suppresses its translation into mutant huntingtin protein—possibly by preventing the ribosome from “reading” the mutated mRNA sequence.

The mismatched siRNAs “are potent, versatile, and selective agents for blocking expression of the mutant huntingtin allele” without affecting the normal one, the researchers write. The agents have nanomolar potencies and more than 30-fold selectivity for the mutant versus the normal gene.

“It’s a potentially important advance,” says professor of medicine Beverly L. Davidson of the University of Iowa, a specialist in inherited genetic diseases of the central nervous system. Corey and coworkers “have shown some clear specificity in in vitro settings,” she adds. “The real proof of the pudding will come when this is tested in vivo, where doses per cell are obviously harder to control. But this is definitely a step in the right direction and gives us an opportunity to extend allele-specific silencing beyond SNP-type approaches. If this can be shown to be functional in vivo, it would be very exciting.”

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