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No one knows what the amyotrophic lateral sclerosis (ALS) gene C9ORF72 does, or how exactly it causes motor neuron disease (MND). No matter—some researchers are speeding ahead to develop and test a potential therapy. ALS researchers, still on a high over the gene’s discovery in 2011 (see ARF related news story), were excited to hear progress reports from two separate collaborations pursuing antisense oligonucleotide therapy at the 23rd annual International Symposium on ALS/MND, held 5-7 December 2012 in Chicago, Illinois. The treatment is based on a theory that mRNAs from mutant C9ORF72 alleles form toxic inclusions, and the antisense oligos should dissolve those poisonous nucleic clots. Michael Baughn of the University of California, San Diego, and Jeffrey Rothstein of Johns Hopkins University in Baltimore, Maryland, described their respective efforts in a poster and oral presentation. They claimed that early results look promising, with the oligos destroying the inclusions and righting abnormal gene expression caused by a hexanucleotide repeat expansion in the C9ORF72 DNA.

Inclusions Confirmed...
The first task, Baughn told Alzforum, was to confirm that C9ORF72 mRNA inclusions exist. Researchers who discovered the genetic association with ALS reported the presence of C9ORF72 mRNA in nuclear foci (Dejesus-Hernandez et al., 2011), but other researchers struggled to replicate the finding. The challenge lay in the repeat's GGGGCC sequence; GC-rich regions are tricky to label because probes pick up other, nonspecific GC-containing strands as well.

Baughn, who works in the laboratory of John Ravits at UCSD, nailed a method to specifically label C9ORF72 foci in collaboration with Clotilde Lagier-Tourenne in the laboratory of Don Cleveland, also at that institution. The trick was to use a locked nucleic acid (LNA) probe. LNA probes are made of DNA with methylene bridges to stabilize the structure in just the right conformation to hook up, specifically, with the C9ORF72 GGGGCC sequence. “It is basically a braced DNA base,” Baughn said. That bracing increases affinity and specificity for the desired target. Researchers aiming to silence the repeat expansion in Huntington’s disease are using a similar technique (see ARF related news story).

With an LNA probe consisting of three hexanucleotide repeats, Baughn and Lagier-Tourenne detected C9ORF72 inclusions. The foci dissolved in the presence of RNase but were resistant to DNase. The inclusions appeared in primary cell lines, including fibroblasts, lymphoblasts, neurons, and glia, isolated from people who had ALS. They were absent in control cells from healthy tissues or from people who had sporadic ALS, familial ALS due to non-C9ORF72 mutations, or Parkinson’s disease.

The foci were mainly nuclear, typically one or two per cell, although occasional cells were jam-packed with inclusions, Baughn said. He and Lagier-Tourenne have not finished characterizing how frequent focus-positive cells are, but Baughn told Alzforum that 9-45 percent of fibroblasts and 22-33 percent of neurons exhibit foci, depending on the cell line they examined.

Simply confirming the presence of C9ORF72 RNA foci was an exciting finding, commented Johnathan Cooper-Knock of the University of Sheffield in the U.K., because their presence only in C9ORF72 repeat-toting cells lends support to the theory that the inclusions are indeed toxic.

“Not only do these foci exist, not only are they composed of RNA, but they are actually pretty common,” Baughn concluded. In spite of the C9ORF72 expansion and foci, the cell lines are fairly healthy, although some grow slowly, he told Alzforum. He was pleased to see the RNA foci were present in fibroblasts and lymphoblasts, because those cell types are easy to grow and would make good models in which to test treatments. Curiously, other researchers have suggested that in approximately one in 15 C9ORF72 patients, the repeat region is shorter in blood than in brain, perhaps making blood cells less than ideal for modeling (see ARF related news story). Baughn used the patient fibroblast lines with the highest rates of RNA inclusions to test an antisense therapy.

…And Denied
For his part, Rothstein, who coauthored one of the C9ORF72 discovery papers (Renton et al., 2011), told Alzforum he and his collaborators started their antisense project the day that manuscript was submitted. They based their idea, in part, on early results with antisense oligonucleotides in fibroblasts and myoblasts from people with myotonic dystrophy type 1, another repeat expansion disorder (reviewed in Mulders et al., 2010).

Rothstein’s group benefited from a ready-made model to test the treatment. He and other researchers have been building a collection of induced pluripotent stem (iPS) cell lines from people with ALS (see ARF related news story), and luckily discovered that one of their lines, from an apparently sporadic case, harbored a C9ORF72 expansion. The researchers made motor neurons and astrocytes from the iPS lines. Like Baughn and Lagier-Tourenne, Rothstein found that RNA inclusions were present, but hardly affected the cells’ health. By profiling the transcriptomes of their lines, the researchers identified some 20 genes encoding secreted proteins that were reliably over- or underexpressed in the C9ORF72 cultures. This signature could eventually make a biomarker for a C9ORF72-targeted therapy, Rothstein said.

Both the Cleveland-Ravits and Rothstein groups teamed up with Isis Pharmaceuticals of Carlsbad, California, which provided candidate antisense oligonucleotides for potentially treating C9ORF72-based ALS. Isis pursues antisense therapies for several neurodegenerative conditions (see ARF related news story on Kordasiewicz et al., 2012, and ARF related news story on Passini et al., 2011). The company is already working on another antisense treatment for ALS, aimed at the ALS gene superoxide dismutase 1 (see ARF related news story). The researchers tried oligonucleotides that bind all C9ORF72 splice forms. In addition, Baughn's group also targeted sequences that appear only in the expanded isoform. Several of the oligonucleotides reduced the frequency of RNA foci, and apparently stabilized gene expression as well.

This evidence from foci-labeling and antisense studies supports the toxic aggregate model. It does not eliminate other possible mechanisms for C9ORF72-based disease. Cells carrying the repeat might suffer haploinsufficiency. For that reason, Baughn and Cooper-Knock are particularly interested in those antisense oligonucleotides that would knock down the abnormal, expanded transcript but leave C9ORF72 mRNA of the proper length alone. “You do not want to create another disease by getting rid of all [of the protein],” Cooper-Knock said.

In an e-mail to Alzforum, John Hardy of University College London, U.K., noted, “This is a hard road. It is a rational approach, but, as with all those antisense [therapies], getting effective delivery will be the challenge.”—Amber Dance.


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News Citations

  1. Corrupt Code: DNA Repeats Are Common Cause for ALS and FTD
  2. Research Brief: Targeting Trinucleotide Repeats Tamps Toxic RNAs
  3. Chicago—RNA Inclusions Offer Therapeutic Target in ALS
  4. Hereditary Diseases: A Natural Fit For iPSC Modeling
  5. “Huntingtin Holiday” Helps Mice Back to Health
  6. Research Brief: Researchers Solicit SMN Understudy to Treat SMA
  7. Chicago—ALS Clinical Trials: New Hope After Phase 3 Setbacks

Paper Citations

  1. . Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron. 2011 Oct 20;72(2):245-56. Epub 2011 Sep 21 PubMed.
  2. . A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron. 2011 Oct 20;72(2):257-68. Epub 2011 Sep 21 PubMed.
  3. . Molecular therapy in myotonic dystrophy: focus on RNA gain-of-function. Hum Mol Genet. 2010 Apr 15;19(R1):R90-7. PubMed.
  4. . Sustained therapeutic reversal of Huntington's disease by transient repression of huntingtin synthesis. Neuron. 2012 Jun 21;74(6):1031-44. PubMed.
  5. . Antisense oligonucleotides delivered to the mouse CNS ameliorate symptoms of severe spinal muscular atrophy. Sci Transl Med. 2011 Mar 2;3(72):72ra18. PubMed.

Other Citations

  1. Read a PDF of the entire series.

Further Reading


  1. . Treatment implications of C9ORF72. Alzheimers Res Ther. 2012 Nov 27;4(6):46. PubMed.
  2. . Co-aggregation of RNA binding proteins in ALS spinal motor neurons: evidence of a common pathogenic mechanism. Acta Neuropathol. 2012 Nov;124(5):733-47. PubMed.