Antisense Therapy Safely Dampens Mutant C9orf72 in ALS Patient

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by Steve Bryson, PhD |

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An experimental antisense oligonucleotide that works to suppress the mutant C9orf72 gene — a cause of amyotrophic lateral sclerosis (ALS) — safely lowered the production of damaging proteins and other molecules in a patient in a pilot trial.

“While other teams have documented that this gene can be suppressed in cells in culture, this is the first time this type of antisense oligonucleotide treatment for C9ORF72 ALS has been demonstrated in a person with ALS,” Robert Brown, MD, the study’s lead author and neurology professor at the University of Massachusetts (UMass) Chan Medical School, said in a university press release.

“The results are very encouraging. It means this is a viable approach to suppressing the mutant C9ORF72 protein that causes most cases of familial ALS,” Brown added.

The study, “Suppression of mutant C9orf72 expression by a potent mixed backbone antisense oligonucleotide,” was published in the journal Nature Medicine.

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Mutations in the C9orf72 gene are the most common genetic cause of ALS, accounting for up to 50% of familial ALS cases and up to 10% of sporadic cases. These mutations consist of too many repeats of six nucleotides — GGGGCC, in which G stands for guanine and C for cytosine, two of the four building blocks of DNA — in the C9orf72 gene sequence.

This gene normally generates three variants of messenger RNA (mRNA) — the molecule derived from DNA that guides protein production — called V1, V2, and V3. Excessive repeats in V1 and V3, however, lead to abnormal proteins, called dipeptide repeat proteins (DPR), that can form toxic protein clumps and contribute to neurodegeneration. These mutated mRNAs can also contribute to RNA foci, or abnormal forms of RNA that build in cells and become toxic.

Antisense oligonucleotides, or ASOs, are short, lab-made, small molecules designed to target specific mRNA molecules and mark them for destruction, ultimately lowering protein production.

“ASOs are essentially anti-messenger RNA agents. Using the genetic sequence you want to target, you can design an antisense oligonucleotide sequence that binds to that mRNA, so the mutant protein never gets made,” said Jonathan Watts, PhD, co-lead author and an associate professor of RNA therapeutics at UMass.

A team led by Watts and Brown designed ASOs to selectively target V1 and V3 while sparing V2, allowing the normal production of C9orf72 protein in the brain and spinal cord.

“We can’t just eliminate all the C9orf72 protein from neurons because that runs a risk of harming the cells,” Watts said. “Any potential treatment needs to be more selective in its targeting.”

The researchers tested five different ASOs in cells derived from an ALS patient who carried C9orf72 gene mutations, with over 1,000 GGGGCC repeats. All five molecules lowered V1 and V3 production to almost undetectable levels in a dose-dependent manner. In addition, the number of cells with RNA foci dropped from 80% to between 20–40%, with fewer foci detected within cells.

Three of these ASOs were discarded after leading to severe seizures in healthy mice. The team then assessed the remaining two (ASO3 and ASO5) in a mouse model of ALS — called C9BAC mice — with about 600 repeats within a human form of the C9orf72 gene. These mice generate DPRs and RNA foci.

After treatment with ASO3 and ASO5, the mice’s brain and spinal cord regions showed a dose-dependent reduction in V1 and V3 repeat-containing mRNAs, as well as fewer dipeptide repeat proteins in brain tissue. Notably, V2 levels were unaffected, and all animals remained healthy after treatment.

Because mice given a higher dose of ASO3 had greater weight loss than other groups, the team used ASO5 in further experiments. They also modified this ASO to create ASO5-2, improving its safety, stability, and distribution in the brain and spinal cord.

ASO5-2 was renamed afinersen by the researchers.

Afinersen’s use safely induced a dose-dependent reduction of DPR proteins, as well as of V1 and V3, for up to 20 weeks (almost five months). Treated C9BAC mice also showed no motor impairments over those months.

“From these observations, we concluded that ASO5-2 can be safely administered to … C9BAC mice via [spinal canal] delivery and that this durably suppresses the offending V1 and V3 transcripts, but not the V2 transcript, of C9ORF72 as well as the toxic [dipeptide repeat proteins],” the researchers wrote.

Its safety was also tested in sheep and monkeys, with no evidence of neurological abnormalities or damage.

A 60-year-old ALS patient with about 2,400 repeats in the C9orf72 gene was then recruited to test afinersen in a single-person pilot study approved by the university’s independent review board and the U.S. Food and Drug Administration.

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The man had recurrent problems with left foot drop while distance running, followed by progressive weakness in his hands and feet.

He also showed enlarged motor neurons — the nerve cells that are damaged in ALS — with evidence of scattered RNA foci. Analysis of the cerebrospinal fluid (CSF) surrounding his brain and spinal cord revealed elevated DPRs.

Three increasing doses of afinersen were given the patient by intrathecal (spinal canal) injection, followed by four additional injections at the highest dose level (2 mg per kg of body weight). No neurological side effects from these injections were reported, and lab safety tests remained within the normal range.

Levels of neurofilament proteins in the CSF — markers of nerve cell damage — progressively rose, peaking after a fourth high dose of afinersen. This rise was transient and the time between doses was extended, the researchers reported.

After the sequence of high doses, his dipeptide repeat protein levels dropped by about 80%, and his ALS functional rating scale (ALSFRS) score generally remained stable, indicating no worsening in disability.

“Our study provides proof of concept that ASO therapy in a human can effectively and safely suppress levels of the C9ORF72 transcript that harbor expansions (V1 and V3) without significantly affecting the predominant V2 transcript,” the researchers wrote.

Findings also “strongly encourage the view that suppressing the expression of the mutant [variants] of C9ORF72 might be clinically beneficial,” they added.

Of note, a Phase 1b/2a clinical trial called FOCUS-C9 (NCT04931862), sponsored by Wave Life Sciences, is evaluating the safety and tolerability of a similar ASO therapy — WVE-004 — in about 50 adults, ages 18 to 80, with ALS or frontotemporal dementia due to C9orf72 mutations. This study is enrolling at sites in Australia, Canada, Ireland, the Netherlands, and the U.K.; information is available here.