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Genomic Therapeutics in Action: Antisense Oligonucleotides Interventions Reverses Neurodegeneration

In This Article

  • Investigators at Massachusetts General Hospital published evidence showing that antisense oligonucleotides (ASOs) treatment may reverse amyotrophic lateral sclerosis (ALS) symptoms in preclinical models
  • ASO therapy addresses aberrant RNA processing and loss of STMN2 function as neurodegenerative hallmarks of TDP-43 proteinopathies
  • ASOs administered into the cerebrospinal fluid of a mouse model rescued STMN2 to normal levels
  • ASO treatment of TDP-43 deficient neurons rescued STMN2 production and restored axon regrowth of injured motor neurons
  • These findings add further credibility to gene and ASO therapy approaches, precisely targeting neurodegenerative disorders

Physician-investigators at Massachusetts General Hospital have recently advanced the field of neurodegenerative disease treatment by delineating the precise mechanisms underlying disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Published in Science, the findings not only enhance our understanding of the etiological intricacies of these diseases but also offer greater precision to apply gene and antisense oligonucleotides (ASO) therapies for targeted interventions to restore normal mRNA and protein levels effectively.

"This study was the culmination of a decade of work to reveal the function of a single protein involved in multiple neurodegenerative diseases," says Clotilde Lagier-Tourenne, MD, PhD, corresponding author of the paper and associate professor of Neurology at Mass General. "The results in preclinical cellular and animal models demonstrate mechanisms directly related to neurological diseases and offer insight into possible therapeutic interventions for these intractable conditions."

A Molecular Hallmark of Multiple Neurodegenerative Diseases

Neurodegeneration associated with ALS is characterized by the progressive loss of motor neurons, eventually leading to paralysis and death. Among ALS cases, 90% occur without a family history (sporadic ALS). Cellular mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43) is implicated in the vast majority of familial and sporadic ALS cases and nearly half of FTD cases, as well as Alzheimer's disease and other neurodegenerative disorders. These specific pathologies are broadly referred to as TDP-43 proteinopathies.

TDP-43 is a nuclear protein that specifically binds thousands of sites in pre-RNA and mRNA that regulate the subsequent translation of proteins. The spectrum of TDP-43 disruptions includes alterations that result in its mislocalization outside the nucleus, aggregation in the cytoplasm of cells, and the loss of numerous proteins that are dependent on TDP-43 function.

As a defining feature of many neurodegenerative disorders, the progressive accumulation of protein aggregates has been a primary research target. However, in the case of TDP-43, Dr. Lagier-Tourenne found that a closer examination of its function in regulating RNA processing revealed the central role of nuclear TDP-43 in sustaining neuron viability and function.

Identifying Critical Pieces in a Complicated Puzzle

While translating genetic DNA into protein, DNA is transcribed into pre-mRNA containing both introns and exons. Exons are the elements that are translated into proteins. Processing pre-mRNA transcripts is performed by proteins such as TDP-43 that bind specific regions and regulate RNA splicing events that remove the introns and join the exons. The result is an mRNA transcript that constitutes a blueprint for the synthesis of a protein.

Stathmin-2 (STMN2) is among many proteins adversely affected by TDP-43 mislocalization. STMN2 is required for axon regeneration and maintenance of motor neuron function, and its loss has been implicated in various neurodegenerative disorders. In 2019, Dr. Lagier-Tourenne and colleagues showed that dysfunctional TDP-43 induces incorrect processing of STMN2 pre-mRNA, ultimately resulting in downregulated STMN2 protein levels.

Specifically, their in vitro findings and ex vivo results using patient-derived cell models of TDP-43 suppression or mutation revealed two key outcomes:

  • Loss of nuclear TDP-43 leads to the production of truncated versions of STMN2 mRNA in cultured cells as well as affected neurons from sporadic and familial ALS patients
  • Reduction in STMN2 impairs the ability of human motor neurons to regenerate their axons after injury

"Our findings highlighted a likely mechanism underlying the role of TDP-43 in these diseases," Dr. Lagier-Tourenne explains. "However, the next step was to identify the mechanisms by which STMN2 loss may contribute to ALS and generate mammalian models to facilitate progress toward an actual therapy."

Generating Preclinical Models of STMN2 Misprocessing

TDP-43 regulates STMN2 levels by binding to specific nucleotide sequences in STMN2 pre-mRNA to prevent alternative splicing and formation of incorrect versions of STMN2 mRNA. TDP-43 does this by blocking access to these areas in pre-mRNA by other factors. To evaluate a possible method to rescue this activity in the absence of TDP-43, Dr. Lagier-Tourenne and her collaborators at the University of California San Diego and at IONIS Pharmaceuticals generated antisense oligonucleotides (ASOs) capable of targeting similar regions in STMN2 pre-mRNA to block alternative-splicing events.

Experiments in TDP-43-deficient human cells subsequently identified ASOs capable of this activity, as evidenced by restored levels of full-length STMN2 mRNA, functional STMN2 protein, and axon regeneration. However, a major hurdle in assessing these ASOs in preclinical models involved differences in the genetic sequence of mouse Stmn2 from that of human STMN2. As a result, mice are not subject to the alternative splicing that can occur in human STMN2 pre-mRNA in the absence of TDP-43.

Addressing this required the creation of "humanized" Stmn2 mice. This was accomplished by incorporating the human STMN2 genetic sequence encoding the problematic region of STMN2 pre-mRNA into the mouse Stmn2 gene. To mimic the disease phenotype in these mice, they altered the TDP-43-binding sequence to promote alternative splicing in Stmn2 pre-mRNA.

After confirming the presence of truncated versions of Stmn2 mRNA and decreased levels of Stmn2 protein in these mice, ASOs were delivered directly to the cerebrospinal fluid (CSF) via intracerebral ventricle injection (ICV). Assessment of their therapeutic efficacy in vivo revealed the following:

  • Up to 50% decreases in truncated versions of Stmn2 mRNA, suggesting that the ASOs successfully inhibited alternative splicing of Stmn2 pre-mRNA
  • Concomitant increases in the percentage of full-length Stmn2 mRNA and increased STMN2 protein levels
  • Recovery of up to 80% of STMN2 protein levels following two ICV injections

Importantly, the findings confirmed that delivery of the ASOs to CSF could result in their uptake by neurons and effective targeting of the correct sites in the humanized Stmn2 pre-mRNA. "These results were critically important to broadening our understanding of how nuclear loss of TDP-43 impacts neuronal function," says Dr. Lagier-Tourenne. "They also demonstrated the feasibility of directly targeting the mechanisms that drive disease progression, which is the first step in creating effective therapies."

An Environment That Fosters Innovative Approaches to Difficult Problems

Dr. Lagier-Tourenne acknowledges the numerous benefits of doing this type of cutting-edge research at Mass General, including longstanding partnerships that enable the acquisition of the unique models necessary for such discoveries. Beyond collaborations with academic and industry partners, she also emphasizes the importance of proximity to clinicians and patients for both research purposes and perspective.

"Our access to patient samples provides immediate feedback and a critical dose of reality against what we see in our models," she says. They also recently started a program that allows research fellows to shadow clinicians and interact with patients. "This experience provides invaluable and long-lasting perspective on the potential impact of their effort in the lab."

Notably, there is precedent for the potential of ASO therapy to treat neurodegenerative disorders. FDA approval of nusinersen to address spinal muscular atrophy was specifically based on a mechanism involving manipulation of pre-mRNA splicing.

Although well-versed in the difficulties in researching neurodegenerative disorders and the limitations presented by various models, Dr. Lagier-Tourenne is motivated by the challenges.

"There's a constant effort to balance the urgency to move things forward with finding the safest and most effective therapeutic option for these devastating diseases that currently have no treatment alternatives," she says. "Each discovery provides momentum, and things are moving faster toward this goal."

The work is a cornerstone for further research that aims to translate fundamental research advancements into clinical interventions, offering not just mechanistic insights but also a pathway for therapeutic advancements, converging discovery from transcriptomic approaches and ASO therapies for proteinopathies.

Learn more about the Healey Center for ALS Research

Learn more about Clotilde Lagier-Tourenne's Lab


Clinical trials of amyotrophic lateral sclerosis (ALS) therapeutic strategies coordinated through Massachusetts General Hospital are prolonging patient life.


Loss of TDP-43 nuclear function is a major event in ALS pathogenesis and leads to widespread RNA processing alterations including reduced stathmin-2 levels. Stathmin-2 is shown to be crucial for axonal regeneration and to represent a novel therapeutic target in ALS.