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Antisense Therapy Against C9ORF72 Looks Promising in ALS Models

Robinson, Richard

doi: 10.1097/01.NT.0000438831.69642.78
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Three new studies — from different labs, and using unique techniques — show that targeting antisense molecules against the gene mutation for C9ORF72 can correct cellular pathology and improve potentially relevant aspects of pathogenesis in cells derived from patients with amyotrophic lateral sclerosis.

Three recent basic science developments — the ability to transform patient fibroblasts into stem cells, the maturing of antisense oligonucleotide therapy, and the discovery of the chromosome 9 open reading frame 72 (C9ORF72) expanded repeat gene mutation — have converged in the field of amyotrophic lateral sclerosis (ALS) to bring potentially meaningful therapy tantalizingly close for a significant number of ALS patients. That convergence is front and center in three new studies from three different laboratories, each showing that targeting antisense molecules against the gene mutation can correct cellular pathology and improve potentially relevant aspects of pathogenesis in cells derived from patients.

Reporting their findings in the Oct. 29 online edition of Proceedings of the National Academy of Sciences (PNAS), John Ravits, MD, and Don Cleveland, PhD, both of the University of California, San Diego, explored the effects of antisense oligonucleotides (ASOs) in patient-derived fibroblasts, and in mice without a mutant gene. In the Oct. 16 Neuron, Jeffrey Rothstein, MD, PhD, and Rita Sattler, PhD, both of Johns Hopkins School of Medicine in Baltimore, began with fibroblasts, and reprogrammed them first into induced pluripotent stem cells (iPS cells) and then into a mixture of neurons and glia. And in the Oct. 23 edition of Science Translational Medicine, Robert H. Baloh, MD, of Cedars-Sinai Medical Center in Los Angeles, also used iPS cells to derive cultures enriched with motor neurons.

“The results from these three studies are very exciting,” said Merit Cudkowicz, MD, chief of neurology at Massachusetts General Hospital, who is a leader in the field of ALS clinical trials and was not involved in any of the new studies. “They all point to the same idea, that reducing the repeat [mutation] may be therapeutic.”

While the new studies overlap in important ways, there are also some potentially significant differences in their results, highlighting just how complex this gene is turning out to be, said Rosa Rademakers, PhD, co-discoverer of the C9ORF72 gene mutation, and associate professor of neuroscience at the Mayo Clinic in Jacksonville, FL. “The papers show there really is hope that knocking down the gene transcript using antisense therapy is a promising approach, but there is not yet one clear picture” of the gene's effects.

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All three studies examined cells derived from ALS patients carrying the expanded C9ORF72 gene mutation, which accounts for at least 25 percent of all familial ALS, and more than 5 percent of sporadic cases. It is also the most common genetic cause of frontotemporal dementia. The normal gene, whose function is unknown, contains fewer than 30 GGGGCC units in the first intron. In the mutant gene, there are hundreds to several thousand of these units. Transcription of the gene forms a tangle of RNA that forms nuclear “foci” reminiscent of the RNA aggregates seen in myotonic dystrophy, which are known to trap RNA binding proteins. Whether a similar mechanism is at work in ALS is an open and urgent question.

All three studies also used ASOs directed at a section of the transcribed RNA, either within the expansion itself or a nearby region, to trigger cellular machinery to destroy the RNA.

In the Science Translational Medicine paper, Dr. Baloh, director of neuromuscular medicine in the department of neurology at Cedars-Sinai, found that the RNA foci co-localized with two RNA binding proteins, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) and Pur-alpha.

In Neuron, Johns Hopkins' Dr. Rothstein, professor of neurology, and Dr. Sattler, assistant professor of neurology, reported that a different binding protein — called adenosine deaminase, RNA-specific, B2 (ADARB2) — was sequestered by foci, and that reducing the protein led to dramatic reduction in the number of foci. That effect mirrors the effect of reducing muscleblind 1 in myotonic dystrophy, Dr. Rothstein noted, suggesting that the RNA-binding protein is required for stabilization of the RNA foci. Reducing ADARB2 also corrected sensitivity to glutamate toxicity, a mechanism implicated in ALS pathogenesis.

Dr. Baloh cautioned that whether any of these proteins are centrally involved in the ALS disease process is not yet clear. “We have a line-up of suspects at this point, and we are going to have to do further experimentation to see if any particular one leads to toxicity.” It could be one of them, or all of them, or other proteins not yet identified, he said.

Since RNA binding proteins in general regulate gene expression, their aberrant interaction with excess RNA has the potential to alter expression of a wide variety of genes, Dr. Rothstein said. All three groups found disease-specific patterns of altered gene expression in the cultured cells, although again with differences among the studies, possibly related in part to the differences between cells. Some gene changes overlapped with those in the motor cortex of patients examined postmortem, but there were significant differences as well.

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If replication is the bedrock of scientific confirmation, then the ability of antisense treatment to reduce RNA foci can be considered confirmed. All three groups dramatically reduced foci using ASOs. Furthermore, both Dr. Baloh and Drs. Rothstein and Sattler found that treatment partially corrected the gene expression changes they saw, and treatment also rescued glutamate susceptibility and neuronal firing abnormalities. Some of the proteins whose production was normalized by treatment are predicted to be secreted into the cerebrospinal fluid, suggesting they may be valuable as biomarkers in future trials, to track whether ASOs are having the desired effect in the nervous system.

Those results are promising, but the PNAS study from Dr. Ravits, professor of clinical neuroscience at the University of California, San Diego, also revealed a new phenomenon in cells containing the mutation: foci of RNA transcribed from the DNA strand opposite to the mutant gene, confusingly enough called the antisense strand. Opposite-strand transcription is not unique to this gene, and may play a role in gene regulation. ASO treatment did not reduce these antisense RNA foci, and did not correct the gene expression changes seen in this study. “If these antisense foci are important pathogenically, they will need to be targeted as well,” Dr. Ravits said.

And then there are the recently discovered repeat-associated non-ATG (RAN) dipeptides, translated from the mutant RNA. Their pathogenic contributions, if any, are unknown, although the phenotypic corrections seen in these studies occurred in the presence of these proteins, suggesting their effects, if any, may be subtle.

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Despite the differences among these three studies, all the authors agreed that the results were promising enough to move ASO treatment for C9ORF72 ALS quickly to clinical trials, assuming toxicology studies do not turn up problems. As an initial test of toxicity, Drs. Ravits and Cleveland depleted C9ORF72 in control mice over several months, and found no overt behavioral or pathologic phenotype.

None of the authors suggested it should be necessary to build a mouse model first to test the therapy. “Once you have a molecular pathway, and you know your drug works in that pathway, you go to humans,” Dr. Rothstein said. Dr. Baloh concurred: “I think we've demonstrated that key features of the human pathology can be recapitulated in a culture dish, and can be reversed by antisense therapy. I think that by itself is enough to say we can move forward.”

“An in vivo model would give us more confidence,” Dr. Cudkowicz said, “but if the model is going to take a couple years to make, I probably wouldn't wait for it. These results are hopeful, and the treatment is right on target for the cause of the disease in these patients.” Genetic testing, she said, is likely to now become a more prominent discussion topic in ALS clinics. “I think we should be testing. We want to find these patients, so that if and when the therapy is ready, we can enroll them in trials.”

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•. Sareen D, O'Rourke JG, Meera P, et al. Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion. Sci Transl Med 2013;5(208):208ra149.
•. Donnelly CJ, Zhang PW, et al. RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention. Neuron 2013;80(2):415–448.
•. Lagier-Tourenne C, Baughn M, Rigo F, et al. Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for amyotrophic lateral sclerosis and frontotemporal dementia. Proc Nat Acad Sci USA 2013; E-pub 2013 Oct. 29.
•. Neurology Today archive on antisense therapy for neurodegenerative disease:
•. PubMed archive on antisense therapy for neurodegenerative disease:
© 2013 American Academy of Neurology