Scientists are taking the offensive against neurodegeneration, with tiny nucleic acids as one of their favored weapons. RNA Metabolism in Neurodegenerative Disease, an SfN satellite conference held October 15-16 in Chicago, hummed with excitement about progress toward antisense oligonucleotide (ASO) therapies that would shut down translation of genes involved in frontotemporal dementia, amyotrophic lateral sclerosis, and Huntington’s disease. “So far all signs are quite positive for ASOs for C9ORF72, SOD1, and huntingtin,” commented Paul Taylor of St. Jude Children’s Research Hospital in Memphis, Tennessee, who co-organized the symposium. Trials are already ongoing or planned for Huntington’s ALS.

ASOs are just one way to get rid of problem RNAs and proteins. Meeting attendees were also intrigued by novel methods to prevent transcription or refold toxic proteins. Aaron Gitler of Stanford University in Palo Alto, California, claimed that a protein required to transcribe trinucleotide and hexanucleotide repeats might make a good drug target. If scientists could suppress it, they might mitigate the production of toxic repetitive RNAs and any downstream protein products, he said. In addition, James Shorter of the University of Pennsylvania Perelman School of Medicine in Philadelphia shared his latest data on augmenting a chaperone that reshapes a variety of malformed proteins involved in neurodegeneration.

Little RNA, big effects.

Robert Brown hopes to use microRNAs to attenuate expression of toxic SOD1 and C9ORF72. [Courtesy of Paul Gardner, University of Canterbury, Christchurch, New Zealand.]

Sensible Strategy
The concept of using ASOs to shut down unwanted protein translation has been around for decades, but finally saw some success in recent years with the 2013 approval of a whole-body ASO treatment, Kynamro®, to treat a genetic cholesterol disorder. Kynamro’s maker, Isis Pharmaceuticals of Carlsbad, California, collaborates with several scientists who hope ASOs will vanquish neurodegenerative disease. Isis’ expertise in designing and manufacturing large quantities of ASOs allows researchers to test hundreds of versions before they pick the best choice for a trial, noted Don Cleveland of the University of California in San Diego. Isis also collaborates with many scientists on preclinical and clinical studies. At the meeting, Cleveland reported on the latest progress for ASOs in animal models as well as human trials.

Cleveland has been working with Isis for 10 years with the goal of ASO treatments for neurodegenerative disease. Some scientists were skeptical about the idea, he told Alzforum, but the researchers determined it was a feasible strategy with a joint UCSD-Isis study of ASOs for the ALS gene SOD1. It slowed down the disease in transgenic mice (see Jul 2006 news). Further, anti-SOD1 ASOs sailed through their first human safety trial with no signs of ill effects (see Jan 2013 conference newsMay 2013 news). Now, the company has a second-generation, more potent SOD antisense oligo to try. It has partnered with Biogen Idec of Cambridge, Massachusetts, to run a Phase 1 study. Biogen is still finalizing the details, a representative told Alzforum, and expects the study to commence in early 2016.

Isis is already in the clinic with a Phase 1/2 trial, started in July, of antisense therapy against huntingtin, the gene mutated in Huntington’s disease. It too delivered promising preclinical results, delaying progression and reversing symptoms in mice (see Jun 2012 news). In the human trial, 36 participants will receive either the ASO or a placebo in a double-blinded fashion, in four infusions over about 13 weeks. Those in the active treatment arm will receive escalating doses. Though safety is the primary outcome measure, the researchers will also assess cognitive performance. Researchers expect the study to wrap up in the fall of 2017.

Cleveland is now targeting C9ORF72, a gene that can contain a large repeat expansion that raises risk for ALS and FTD. The scientists designed an ASO that targets only the repeat-containing RNA, and confirmed that it left the normal gene unperturbed. “This is almost allele-selective silencing, the sort of Holy Grail in these kinds of gene-silencing approaches,” Cleveland said. Cleveland, along with Clotilde Lagier-Tourenne of Massachusetts General Hospital in Charlestown, tested the antisense therapy in new mouse models of C9ORF72 toxicity they developed (see Part 1 of this series). They treated four to seven mice with a single ASO injection into the brain’s ventricles. Two and four weeks later, when the researchers sacrificed the mice, the animals had fewer repeat-containing mRNAs in the brain and spinal cord than mice treated with control, ineffective ASOs. The treatment also reduced levels of at least two of the poly-dipeptides encoded by those repeats, poly-glycine-proline and poly-glycine-alanine. The researchers have not yet measured the other peptides. They plan to check if the treatment alleviates learning defects in these mice, as well, said Jie Jiang of Cleveland’s lab, who presented the project in a poster. Cleveland predicted C9ORF72 ASOs would be ready for a human trial in early 2017.

Robert Brown of the University of Massachusetts Medical School in Worcester prefers a different approach to rid neurons of unwanted RNAs. MicroRNAs are natural, noncoding sequences that bind to target mRNAs to fine-tune gene expression, by either preventing translation or promoting mRNA degradation. Brown and colleagues have already engineered an artificial miRNA against SOD1, which they packaged into an adenoassociated virus vector. They injected the vector into the spinal cord of two-month-old transgenic mice expressing a human SOD1 with the glycine-93-alanine mutation that causes ALS. The virus delivered that miRNA gene to the nucleus, so the neurons generated the miRNA themselves after the one-time treatment. It knocked down the mutated gene and slowed the disease (Wang et al., 2014). Now, the lab is using the same strategy to suppress C9ORF72.

Brown’s group has tried the miRNAs on cultured cells, and also provided them via virus to a mouse model they engineered to overexpress the repeat-laden human C9ORF72 gene. The researchers are experimenting with intrathecal, intraventricular, and even intravenous delivery, he said, since some viruses can cross the blood-brain barrier. They have treated both newborns and adults. “We see promising results,” Brown told Alzforum. The repeat RNAs formed fewer potentially toxic foci, and expression of poly-dipeptides fell.

How do Brown’s microRNAs compare to Cleveland’s ASOs? Each has its pluses and minuses, Brown said. Unlike viruses, most ASOs cannot cross the blood-brain barrier. AAV delivers a permanent transgene, so it would require only one treatment as opposed to regular infusions of ASOs. However, that means there would be no way to take back a viral microRNA treatment gone awry. Brown also noted that microRNAs mature in the nucleus and then find their target mRNAs in the cytoplasm. Scientists recently determined that C9ORF72 repeats inhibit nucleocytoplasmic transport, which Brown speculated might interfere with the transport of miRNAs to the cytoplasm (see Aug 2015 news). Brown’s group is exploring whether traffic jams at the nuclear membrane will interfere with their microRNAs, he told Alzforum.

Transcription Factor
In the case of C9ORF72, treatment with ASOs and miRNAs faces another complication. Because the cell transcribes the repeats in both the sense and antisense directions, oligonucleotides specific for the forward and backward directions may be needed to treat the disease (see Nov 2013 news). Gitler proposed a potential solution that would eliminate both antisense and sense transcripts at once.

A few years ago, scientists discovered that the yeast transcription elongation factor SPT4 assisted RNA polymerase in copying lengthy trinucleotide repeats such as those found in mutant huntingtin (see Feb 2012 news). SPT4—and its mammal homolog Supt4h—help the polymerase stay attached to the repetitive DNA, but are not essential for most transcripts or cell viability (Malone et al., 1993). Since then, the same group has knocked down Supt4h in HD model mice, and found it diminished expression of huntingtin RNA and protein (Cheng et al., 2015). Might SPT4 also assist RNA polymerase transcribing C9ORF72 hexanucleotide repeats? Gitler’s group found that if they eliminated SPT4 from yeast, C9ORF72 repeats were no longer transcribed or translated. He plans to test if downregulating Supt4h protects cell lines derived from people with C9ORF72 expansions, or mice that model C9ORF72 toxicity.

Optimized Chaperone
While Gitler, Brown, and Cleveland have prevention of translation in mind, Shorter has devised a solution to problem proteins once they are out and about, and misfolded as toxic neurodegeneration proteins often are. The researchers started with Hsp104, a chaperone known to disassemble oligomers. Using error-prone PCR, they generated several Hsp104 variants that protect yeast from toxic proteins. The modified proteins untangled and refolded mutant forms of α-synuclein, TDP-43, FUS, and TAF15 into their proper conformations (see Aug 2014 news). At the meeting, Shorter added to that list the ribonucleoprotein coded by the ALS gene hnRNPA2, and a poly-dipeptide (poly-proline-arginine) encoded by C9ORF72 repeats. “These [chaperones] are the only things we know about that are able to separate aggregated proteins and recover native forms,” Shorter said, adding he would love to do the same in people with ALS.

Shorter shared some preliminary data to suggest that chaperones can act as disaggregases in animals. With his modified Hsp104, he was able to dissolve aggregated SOD1 in nematodes and globs of FUS in cultured human fibroblasts, he said. He has yet to test the modified chaperone in mice. While people have no Hsp104 homolog, Shorter speculated that he might be able to modify a different human chaperone to make it disaggregate toxic proteins. He envisions either providing the protein as a therapeutic, or the gene via gene therapy. The idea of disassembling aggregates has appeal, commented Robert Bowser of the Barrow Neurological Institute in Phoenix. However, he added, any such therapeutic would likely require tight control so it would not unwind essential proteins.

Cleveland hinted he is already looking ahead to the next big thing, which he predicted would be CRISPR-based gene editing (see Sep 2014 series). “You could even imagine getting gene correction,” he said. Brown told Alzforum exciting times are ahead. “Gene silencing and gene replacement are finally coming of age.”—Amber Dance


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News Citations

  1. Treatment Trends: Tapping Stem Cells, DNA, and RNA to Save Neurons
  2. Chicago—ALS Clinical Trials: New Hope After Phase 3 Setbacks
  3. Paper Alert: Antisense Oligonucleotide Therapy Safe for ALS?
  4. “Huntingtin Holiday” Helps Mice Back to Health
  5. C9ORF72 Mice Point to Gain of Toxic Function in ALS, FTD
  6. ALS Gene Repeats Obstruct Traffic Between Nucleus and Cytoplasm
  7. Sense, Antisense: C9ORF72 Makes Both Forms of RNA, Peptides
  8. Huntington’s Strategies Tap Transcription, Htt Phosphorylation
  9. Yeast Chaperone Melts Protein Aggregates

Series Citations

  1. CRISPR: A New Tool For Gene Editors

Paper Citations

  1. . Widespread spinal cord transduction by intrathecal injection of rAAV delivers efficacious RNAi therapy for amyotrophic lateral sclerosis. Hum Mol Genet. 2013 Oct 9; PubMed.
  2. . Molecular and genetic characterization of SPT4, a gene important for transcription initiation in Saccharomyces cerevisiae. Mol Gen Genet. 1993 Mar;237(3):449-59. PubMed.
  3. . Effects on murine behavior and lifespan of selectively decreasing expression of mutant huntingtin allele by supt4h knockdown. PLoS Genet. 2015 Mar;11(3):e1005043. Epub 2015 Mar 11 PubMed.

External Citations

  1. Isis Pharmaceuticals
  2. safety trial
  3. Phase 1/2 trial
  4. C9ORF72

Further Reading


  1. . Identification and Characterization of Modified Antisense Oligonucleotides Targeting DMPK in Mice and Nonhuman Primates for the Treatment of Myotonic Dystrophy Type 1. J Pharmacol Exp Ther. 2015 Nov;355(2):310-21. Epub 2015 Sep 1 PubMed.
  2. . Gene suppression strategies for dominantly inherited neurodegenerative diseases: lessons from Huntington's disease and spinocerebellar ataxia. Hum Mol Genet. 2016 Apr 15;25(R1):R53-64. Epub 2015 Oct 26 PubMed.
  3. . Antisense Oligonucleotide Therapy for the Treatment of C9ORF72 ALS/FTD Diseases. Mol Neurobiol. 2014 May 9; PubMed.
  4. Antisense-mediated Exon Skipping Decreases Tau Protein Expression: A Potential Therapy for Tauopathies. Mol Ther Nucleic Acids. 2014 Oct 21;3:e204. PubMed.
  5. . Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration. Proc Natl Acad Sci U S A. 2013 Nov 19;110(47):E4530-9. Epub 2013 Oct 29 PubMed.
  6. . Antisense Reduction of Tau in Adult Mice Protects against Seizures. J Neurosci. 2013 Jul 31;33(31):12887-97. PubMed.
  7. . Antisense oligonucleotides: treating neurodegeneration at the level of RNA. Neurotherapeutics. 2013 Jul;10(3):486-97. PubMed.