The stutter that causes an overabundance of DNA repeats in diseases such as Huntington’s is tricky to silence because the normal genes, too, have their own, smaller share of repeats. Scientists may have found a way out of this conundrum with RNA interfering technology that preferentially binds longer repeats. In a paper published online May 3 in Nature Biotechnology, researchers from the University of Texas Southwestern Medical Center in Dallas describe how modified nucleic acids specifically dampen translation of the longer, mutant form of huntingtin, the gene that causes Huntington disease. The technique, though in the early stages of development, could potentially apply to 19 diseases that are caused by similar DNA repeats.

The normal huntingtin gene contains up to 35 repeats of the trinucleotide CAG, encoding glutamine; the disease-causing form contains more than 35, up to and beyond 100 repeats. “The challenge is to inhibit the mutant allele, but not the wild-type,” said principal investigator David Corey, who worked with first author Jiaxin Hu and colleagues on the project. RNA-silencing techniques that target all forms of huntingtin risk side effects by destroying normal transcripts. Other researchers have developed siRNAs that target specific huntingtin alleles, based on single nucleotide polymorphisms (Pfister et al., 2009), but treatment would require a panel of such oligonucleotides tailored for each genotype. Corey’s concept is potentially one-size-fits-all, or at least one-size-fits-most.

Hu and colleagues engineered peptide nucleic acids (PNAs), which have normal nucleotides on a peptide-based backbone, and RNAs seeded with occasional locked nucleic acids (LNAs)—ribonucleotides trapped in a conformation that makes them more likely to bind complementary strands. These molecules preferentially bound the longer forms of huntingtin transcripts in cell culture, probably because of structural differences between the long and short mRNAs, Corey said. The most selective PNAs blocked all translation of mutant mRNAs while allowing the wild-type to express at normal levels. The oligonucleotide treatment also decreased the sensitivity of mutant huntingtin-expressing cells to glutamate toxicity: untreated cells exposed to glutamate had 70 percent apoptosis, but the oligos reduced apoptosis to 30 percent, close to the rate wild-type cells suffer in the presence of glutamate.

“It was somewhat unexpected that it worked as well as it did,” said Frank Bennett of Isis Pharmaceuticals in Carlsbad, California, which is collaborating with Corey. “It is binding a sequence that is common between both the wild-type and the mutant variant of huntingtin…I was somewhat surprised that there was this much discrimination.”

Via molecular tinkering, such as attaching an additional peptide, the researchers were able to increase selectivity for the disease form of huntingtin. “It told us that in the whole world of chemical species, there is something out there that can be very selective,” Corey said. He intends to zero in on the most selective molecule in future experiments.

In the current study, a peptide-conjugated PNA with 19 CAG trinucleotide repeats was the most selective, but LNAs are a better bet for clinical applications because they are already in use in trials, Corey said. “People know how to make [LNAs] on a large scale, they know what the toxicology is, and they are familiar with administering it to patients. That is a big advantage to us,” he said.

The researchers also demonstrated that their technique was effective in blocking mutant ataxin-3 mRNA, another CAG-repeat gene that causes Machado-Joseph disease, suggesting the therapy could translate to other trinucleotide repeat conditions.

“I think this is a step in the right direction,” said Beverly Davidson of the University of Iowa in Iowa City, who was not involved in the current study. “The demonstration of efficacy in vitro was very promising.” The next step, naturally, will be to try the oligonucleotides in mice.—Amber Dance


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Paper Citations

  1. . Five siRNAs targeting three SNPs may provide therapy for three-quarters of Huntington's disease patients. Curr Biol. 2009 May 12;19(9):774-8. PubMed.

Further Reading


  1. . Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits. Proc Natl Acad Sci U S A. 2007 Oct 23;104(43):17204-9. PubMed.
  2. . RNAi therapy for neurodegenerative diseases. Curr Top Dev Biol. 2006;75:73-92. PubMed.
  3. . Designing siRNA that distinguish between genes that differ by a single nucleotide. PLoS Genet. 2006 Sep 8;2(9):e140. PubMed.
  4. . RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model. Proc Natl Acad Sci U S A. 2005 Apr 19;102(16):5820-5. PubMed.
  5. . RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat Med. 2004 Aug;10(8):816-20. PubMed.
  6. . Molecular architecture of CAG repeats in human disease related transcripts. J Mol Biol. 2004 Jul 16;340(4):665-79. PubMed.

Primary Papers

  1. . Allele-specific silencing of mutant huntingtin and ataxin-3 genes by targeting expanded CAG repeats in mRNAs. Nat Biotechnol. 2009 May;27(5):478-84. PubMed.