Article in brief

An experimental treatment given to a marathon runner in the early stages of ALS resulted in a significant and long-lasting reduction in levels of repeatedly toxic dipeptide proteins in cerebrospinal fluid. The treatment, an antisense oligonucleotide aimed at suppressing a repeat expansion in the non-coding region of chromosome 9 open reading frame 72, or C9ORF72, has not yet demonstrated clinical benefit, but the patient’s condition has since remained stable. the infusion. therapeutic protocol began in 2019.

An antisense oligonucleotide (ASO) designed to suppress a repeat expansion in the noncoding region of chromosome 9 open reading frame 72, or C9ORF72, in a patient with the early stages of amyotrophic lateral sclerosis (ALS) led to significant and long-lasting reduction in toxic dipeptide protein (DPR) levels in cerebrospinal fluid.

DPRs are produced and aggregated in people with C9ORF72 mutations. The results of the experimental treatment and the laboratory and animal studies leading to its use in this patient were published in December in natural medicine.

ASO, developed at the University of Massachusetts RNA Therapeutic Institute, had been tested in the laboratory on transgenic mice with C9ORF72 pathology, as well as on sheep and monkeys before the first human patient received it. individualized experimental treatment. It is impossible to say if there is a clinical benefit, but the study’s principal investigator, Robert H. Brown, MD, DPhil, director of the neurotherapy program, said the patient’s condition remained stable. since the start of the infusion therapy protocol in 2019. .

“It’s a new era for ALS patients,” said Dr. Brown. Other ASO studies have shown that the product of mutations can be suppressed, but it’s still unclear if it will ultimately have clinical benefit for patients, he added. “The next step is to start a clinical trial on several people to see if this treatment can slow the progression of the disease.”

Repeated expansions of the C9ORF72 gene in ALS and frontotemporal dementia were only identified in 2011 and its regulatory role in the brain is still under investigation. C9ORF72 mutations are the most common genetic cause of ALS and frontotemporal dementia, seen in approximately 40% of familial ALS cases and 7% of sporadic cases. These mutations can also cause about 25% of familial cases of FTD.

So far, studies have shown that the gene product is essential for vesicle trafficking and the regulation of systemic and neural inflammation. People normally have fewer than 20-30 repeats in the gene sequence, but patients with one mutated copy (the other allele is normal) may have hundreds or even thousands of repeat expansions. Repeated expansion leads to partial loss of the C9ORF72 protein and produces neurotoxic forms of the DPR proteins. The hope is that ASO treatment will reduce these toxic proteins and prevent neurons from dying. Dr. Brown said the team will seek federal approval to treat other ALS patients with the C9ORF72 mutation. In all models tested so far, and in the patient, the drug appears to be safe, Dr. Brown said.

ASOs act by binding to messenger RNA and inducing degradation of the transcript, preventing synthesis of the abnormal protein.

Study details

Dr. Brown collaborated with Jonathan Watts, PhD, a chemist who did his post-doctorate in pharmacology and biochemistry and has worked at the RNA Therapeutic Institute for three years. They have conducted expanded access clinical studies in ALS.

Dr. Watts studied the structure of the C9ORF72 gene and worked on ways to silence its mutated form which contained the repeats and to test whether it could reduce the toxic forms of the poly(GP) dipeptides.

Dr. Watts and his chemists chemically synthesized the ASOs, then used cells derived from C9-ALS/FTD patients and a C9ORF72 BAC transgenic mouse to test the ASOs in these studies. They focused on two specific isoforms of the C9ORF72 gene that generate the toxic DPRs and several ASOs knocked down levels of the toxic protein. ASOs selectively turn off the expression of transcripts containing G4C2 repeats by binding to messenger RNA so that the mutant protein is never made.

After tests in mouse fibroblasts and neurons showed that ASO suppresses C9ORF72, the researchers conducted studies in two transgenic mouse models, finding a dose-dependent reduction of approximately 80% in both transcripts ( V1 and V3) which were important to silence the generated messages. repeated expansions. The animals also had a sustained 90% reduction in DPR levels in the cerebrospinal fluid. They moved on to larger animals – sheep and monkeys – to test its safety.

Once the data showed ASO was safe and could be targeted to the right cells to suppress the repeated expansion of the genetic code, investigators identified one patient for individualized treatment, a 60-year-old marathon runner. who had developed left foot droop, followed by leg and foot weakness. The C9ORF72 mutation was part of his family heritage; several of his relatives had succumbed to ALS. This patient has about 2,400 repeat expansions on his C9ORF72 gene. With no approved treatment in sight, he enrolled in the study. A basic lumbar puncture showed elevated levels of DPR.

ASO (0.5 mg kg) was injected into his spinal canal in August 2019, followed again two weeks later by another slightly higher intrathecal dose (1.0 mg kg) than the first. In January 2020, he received a third dose at 1.5 mg kg and a month later a fourth dose at 2.0 mg kg. This same dose was administered four more times over the following months.

Dr Brown said the patient had no medical or neurological adverse effects and laboratory safety studies also showed no concerning signs. (Investigators reported very mild pleocytosis on the fourth and fifth CSF scans. They also observed a gradual increase in phosphorylated neurofilament heavy and light chains, which peaked after the fourth dose and then partially abated after extended the interval between doses.) DPR levels in his CSF dropped by about 80%, which suggested to the scientists that they were able to effectively silence transcripts containing repeats.

The Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS), used to measure disease progression, was largely stable. Initially, it was 38 (48 is normal), decreasing to 33 but then improving to 38 as doses of ASO increased, Dr. Brown said.

Expert commentary

“The goal of using this antisense oligonucleotide technology is to treat ALS caused by the repeated expansion of C9ORF72 by removing toxic bRNA and thereby provide some clinical benefit,” said Neil Shneider, MD. , PhD, Claire Tow Associate Professor of Motor Neuron Disorders and Director of the Eleanor and Lou Gehrig ALS Center at Columbia University Vagelos College of Physicians and Surgeons. “ASO given to this patient has been shown to significantly reduce DPRs in the cerebrospinal fluid. It’s impressive. But in what interest will this modify the evolution of the disease? We just don’t know, and the authors were very careful not to make any claims of effectiveness.

Dr. Shneider said experts continue to debate how repeated expansion of C9ORF72 causes these two neurodegenerative diseases. There are several possible theories: repeated expansion could interrupt expression of the gene product and cause loss of function leading to neurodegeneration; the mRNA-reading machinery makes proteins on all frames of both strands of the C9 expansion, producing five DPRs that are neurotoxic; or RNA can sequester other RNA-binding proteins, disrupting their normal function. Or it could be any or all of these factors.

“No matter the mechanism, we want to get rid of this toxic RNA,” Dr. Shneider said.

“Patients need to understand that this is an experimental treatment,” said Annemieke Aartsma-Rus, PhD, professor of translational genetics in the department of human genetics at Leiden University Medical Center in the Netherlands. “We are in the same field and use the same tools to achieve different things. Boston scientists want to break down gene transcription. We want to manipulate gene transcripts to restore a missing protein. They developed ASOs for one patient, but the hope is that it would work for other ALS patients.

“The challenge is that ALS is progressive and by the time they can enter a clinical trial it may be too late. Time is running out for these patients. We say time is neurons and every day, patients lose neurons that have disappeared irreversibly.

Dr Aartsma-Rus added: “It is not known whether ASO works to slow down the disease process, but a biomarker – the dipeptide repeat protein – descends into the CSF and the patient’s clinical picture has remained stable ever since. that he started treatment. But we don’t know what would have happened if he hadn’t received the treatment. This sounds encouraging, but we cannot conclude that it will benefit him clinically. »

Disclosures

The authors of the study have filed a patent related to this research. Dr. Brown is co-founder of Apic Bio.